Humanized antibodies to ganglioside GM2

ABSTRACT

Chimeric human antibody expression vectors are constructed by inserting the antibody heavy chain variable region-encoding cDNA and antibody light chain variable region-encoding cDNA isolated from hybridomas producing a mouse or rat monoclonal antibody reacting with the ganglioside GM 2  respectively into an expression vector for use in animal cells which contains the human antibody heavy chain constant region- or human antibody light chain constant region-encoding cDNA. The expression vectors are introduced into animal cells and the transformant thus obtained is cultured for the production of a chimeric human antibody reacting with the ganglioside GM 2 . In contrast to mouse monoclonal antibodies, the chimeric human antibodies of the invention will not cause anti-mouse immunoglobulin antibody production in the patient&#39;s body but shows a prolonged blood half-life, with a reduced frequency of adverse effects, so that it can be expected to be superior to mouse monoclonal antibodies in the efficacy in the treatment of human cancer, for instance.

This is a continuation-in-part of application Ser. No. 08/483,528, filedJun. 7, 1995, which is a continuation-in-part of application Ser. No.08/116,778, filed Sep. 7, 1993, now U.S. Pat. No. 5,830,470 the contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to humanized antibodies reacting with theganglioside GM₂. The humanized antibodies do not cause production ofanti-mouse immunoglobulins in the patient's body as compared with mousemonoclonal antibodies, hence the incidence of adverse effects possiblycaused by them is much lower, their blood half-lives are longer and,further, their anti-tumor effector effect is greater. Therefore, thehumanized antibodies are expected to produce improved therapeuticeffects as compared with mouse monoclonal antibodies.

BACKGROUND OF THE INVENTION

When administered to humans, mouse antibodies are generally recognizedas foreign matters, inducing production of anti-mouse immunoglobulinantibodies in the human body. It is known that the former antibodiesreact with the latter antibodies to produce adverse effects [J. Clin.Oncol., 2, 881 (1984); Blood, 65, 1349 (1985); J. Natl. Cancer Inst.,80, 932 (1988); Proc. Natl. Acad. Sci. U.S.A., 82, 1242 (1985)] and thatthe mouse antibodies undergo rapid clearance [J. Nucl. Med., 26, 1011(1985); Blood, 65, 1349 (1985); J. Natl. Cancer Inst., 80, 937 (1988)],thus showing only a reduced efficacy [J. Immunol., 135, 1530 (1985);Cancer Res., 46, 6489 (1986)]. Attempts have been made to solve theseproblems by deriving, from mouse monoclonal antibodies, chimeric humanantibodies or CDR (complementarity determining region)-transplantedantibodies (reshaped antibodies) using gene engineering technique. In ahuman chimeric antibody, the variable regions thereof are of mouseorigin and the constant regions thereof are of human origin [Proc. Natl.Acad. Sci. U.S.A., 81, 6851 (1984)] and it is reported that whenadministered to humans, said antibody causes litte human anti-mouseimmunoglobulin antibody production, its blood half-life being 6-foldlonger [Proc. Natl. Acad. Sci. U.S.A., 86, 4220 (1989)]. The CDR-graftedantibodies are antibodies resulting from replacement of the CDRs in ahuman antibody alone with the CDRs from an animal other than the human[Nature, 321, 522 (1986)] and, in an experiment with monkeys, suchantibodies showed reduced immunogenicity and 4- to 5-fold higher serumhalf-lives as compared with mouse antibodies [J. Immunol., 147, 1352(1991)].

As regards the cytocidal activity of antibodies, it is reported that theFc region of a human antibody is more potent in activating humancomplement and human effector cells than the Fc region of a mouseantibody. Thus, for instance, a chimeric antibody derived from a mousemonoclonal antibody to the ganglioside GD₂ and containing a humanantibody Fc region enhances the human effector cell-mediated antitumoreffect [J. Immunol., 144, 1382 (1990)]. Similar results are reported forCDR-grafted antibodies [Nature, 332, 323 (1988)]. Such results indicatethat, for clinical use, humanized monoclonal antibodies are preferred tomouse monoclonal antibodies.

The antibody classes include IgA, IgM, IgG, IgD and IgE and , in mice,the class IgG includes four subclasses, namely IgG₁, IgG₂ a, IgG₂ b andIgG₃ (in humans, IgG₁, IgG₂, IgG₃ and IgG₄). When antigens areadministered to animals, the antibodies produced mostly belong to theclasses IgM or IgG. IgG molecules have a molecular weight of about160,000 daltons and a dimeric structure and are relatively easy tohandle. IgM molecules are large with a molecular weight of about 900,000daltons and occur in the form of a complicated pentameric structurecoupled with the joining (J) chain, hence they have the followingdrawbacks: they are difficult to purify; they tend to agglutinate, henceare difficult to store; they are readily inactivated by partialdecomposition in the presence of a protease, hence it is difficult toprepare Fab fragments; and they lose their binding activity in manyinstances upon chemical modification, for example chemical binding of ananticancer agent or a toxin [J. W. Goding: Monoclonal Antibodies:Principles and Practice, Academic Press, 1986]. As to which are superiorin therapeutic effect against cancer, IgG class monoclonal antibodies orIgM class monoclonal antibodies, reference may be made to a detailedstudy made by Bernstein et al. using an IgG class monoclonal antibodyand an IgM class monoclonal antibody to the lymphocyte Thy-1 antigen[Monoclonal Antibodies, edited by R. H. Kennet, T. J. Mckearn and K. B.Bechtol, Plenum Press, 1980, p. 275]. According to the reference, an IgGclass monoclonal antibody and an IgM class monoclonal antibodycomparable in terms of reactivity to Thy-1 antigen-positive lymphocytes,were compared in terms of antitumor effect. While the IgM monoclonalantibody was superior in in vitro complement-dependent antitumor effect,the IgG class monoclonal antibody showed a significant antitumor effectin in vivo antitumor effect in cancer-bearing mice, with no antitumoreffect being observed with the IgM class monoclonal antibody. It wasfurther revealed that, as compared with the IgG class monoclonalantibody, the IgM class monoclonal antibody showed a very shorthalf-life in the blood after administration, in an isotope-labeled form,to mice. These results indicate that the monoclonal antibodies to beused clinically in humans should preferably be of the IgG class.

Gangliosides, a class of glycolipids, are constituents of animal cellmembranes. These molecules are composed of a carbohydrate chain, whichconstitutes a hydrophilic side chain, and sphingosine and a fatty acid,which constitute hydrophobic side chains. It is known that theganglioside species expressed and the amount thereof differ between cellspecies, organ species, and animal species, among others. Furthermore,it has been reported that the ganglioside expressed changedquantitatively and qualitatively during the process of cancerdevelopment [Cancer Res., 45, 2405 (1985)]. For example, expression ofthe gangliosides GD₂, GD₃ and GM₂ has been reported in neuroblastoma,lung small cell carcinoma, and melanoma, which are highly malignantneural ectodermal tumors [J. Exp. Med., 155, 1133 (1982); J. Biol.Chem., 257, 12752 (1982); Cancer Res., 47, 225 (1987); ibid., 47, 1098(1987); ibid., 45, 2642 (1985); Proc. Natl. Acad. Sci. U.S.A., 80, 5392(1983)].

GM₂, one of the gangliosides that are sialic acid residue containingglycolipids, occurs only in trace amounts in normal cells but is foundin increased amounts in cancer cells in lung small cell carcinoma,melanoma, neuroblastoma, etc. Monoclonal antibodies to GM₂ areconsidered to be useful in the treatment of such cancers [Lancet, 4, 786(1989)]. However, those monoclonal antibodies to GM₂ that have so farbeen reported are of the human IgM class or of the rat IgM, mouse IgM ormouse IgG class [Cancer Res., 46, 4116 (1986); Proc. Natl. Acad. Sci.U.S.A., 79, 7629 (1982); Cancer Res., 48, 6154 (1988); J. Biol. Chem.,264, 12122 (1989)].

Anti-ganglioside GM₂ monoclonal antibodies, if produced in the form ofhumanized antibodies, for example chimeric human antibodies orCDR-grafted antibodies, which are not expected to induce anti-mouseimmunoglobulin antibody production in the patient's body, producereduced adverse effects and show a prolonged blood half-life and anenhanced antitumor effector effect. These antibodies are thus expectedto be superior in therapeutic effect to the corresponding mousemonoclonal antibodies.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide humanized antibodiesto the ganglioside GM₂ (hereinafter, "humanized anti-GM₂ antibodies")which are useful in the treatment of cancers of neural ectodermalorigin, among others.

The present inventors prepared the antibody heavy chain (hereinafter, "Hchain") variable region (hereinafter "V_(H) ") cDNA and light chain(hereinafter, "L chain") variable region (hereinafter, "V_(L) ") cDNAsfrom mRNAs isolated from the hybridomas KM750 and KM796, described inEP-A-0 508 472. These hybridomas produce IgG₃ class mouse monoclonalantibodies to the ganglioside GM₂. V_(H) and V_(L) cDNAs were alsoprepared from mRNAs isolated from the hybridoma KM603, which produces anIgM class rat monoclonal antibody to the ganglioside GM₂. Chimeric humanantibody expression vectors were constructed by inserting the cDNA intoan expression vector containing human antibody H chain constant region(hereinafter, "C_(H) ") or human antibody L chain constant region(hereinafter, "C_(L) ") encoding sequences. Such vectors were thenintroduced into animal cells to effect the production ofanti-ganglioside GM₂ chimeric human antibodies. Among the chimericantibodies produced, the anti-ganglioside GM₂ chimeric human antibody,KM966, was found to react with the ganglioside GM₂ and show cytocidalactivity. The H chain variable region of KM966 contains an amino acidsequence segment as defined by SEQ ID NO:1 and includes the 1st to 120thamino acids of that sequence and the L chain variable region of KM966contains an amino acid sequence segment as defined by SEQ ID NO:2 andincludes the 1st to 107th amino acids of said sequence. The presentinvention is based, at least in part, on these findings.

The present invention thus relates to a humanized antibody reacting withthe ganglioside GM₂.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates plasmids, pKM796H1 and pKM796L1.

FIG. 2 illustrates plasmids, pKM750H1 and pKM750L1.

FIG. 3 illustrates plasmids, pKM603H1 and pKM603L1.

FIG. 4 shows a construction scheme for a plasmid, pAGE147.

FIG. 5 shows a construction scheme for a plasmid, pAGE148.

FIG. 6 shows a construction scheme for plasmids, pChi796HM1 andpChi750HM1.

FIG. 7 shows a construction scheme for plasmids, pChi796HMS1 andpChi750HMS1.

FIG. 8 shows a construction scheme for plasmids, pChi796LI1 andpChi750LI1.

FIG. 9 shows a construction scheme for plasmids, pChi796LM1 andpChi750LM1.

FIG. 10 shows a construction scheme for plasmids, pChi796LMS1 andpChi750LMS1.

FIG. 11 shows a construction scheme for plasmids, pChi796H107 andpChi750H107.

FIG. 12 shows a construction scheme for plasmids, pChi796HL1 andpChi750HL1.

FIG. 13 shows a construction scheme for a plasmid, pChi603HM1.

FIG. 14 shows a construction scheme for a plasmid, pChi603HMS1.

FIG. 15 shows a construction scheme for a plasmid, pChi603LI1.

FIG. 16 shows a construction scheme for a plasmid, pChi603LM1.

FIG. 17 shows a construction scheme for a plasmid, pChi603LMS1.

FIG. 18 shows the electrophoretic patterns in SDS-PAGE (using 4-15%gradient gels) of purified chimeric human anti-GM₂ antibodies, KM966 andKM967. The patterns obtained under reducing conditions are shown on theleft side and those obtained under nonreducing conditions on the rightside. From the left, the lanes include low molecular weight markers,KM967 and KM966 (reducing conditions), and KM967 and KM966 (nonreducingconditions).

FIG. 19 shows the electrophoretic patterns in SDS-PAGE (using 4-15%gradient gels) of a purified chimeric human anti-GM₂ antibody, KM968.The pattern obtained under reducing conditions is shown on the left sideand that obtained under nonreducing conditions on the right side. Fromthe left, the lanes include high molecular weight markers, low molecularweight markers, a standard human IgG, KM968 (reducing conditions), thesame low molecular weight markers, the standard human IgG, and KM968(nonreducing conditions).

FIG. 20 graphically shows the CDC (complement dependent cytotoxicity)activities of KM966 against the human lung small cell carcinoma celllines SBC-3 and LU-135. The ordinate indicates the cytotoxic activityand the abscissa the concentration of the antibody added. The solid barsindicate the CDC activities of KM-696 and the shaded bars the CDCactivities of KM966.

FIG. 21 graphically shows the CDC activities of KM966 against the humanlung squamous cell carcinoma cell line PC-10 and human lungadenocarcinoma cell line RERF-LC-MS. The ordinate indicates thecytotoxicity and the abscissa the concentration of the antibody added.The solid bars indicate the CDC activities of KM-696 and the shaded barsthe CDC activities of KM966.

FIG. 22 graphically shows the CDC activities of KM966 against the humanlung large cell carcinoma cell line PC-13 and human neuroblastoma cellline NAGAI. The ordinate indicates the cytotoxic activity and theabscissa the concentration of the antibody added. The solid barsindicate the CDC activities of KM-696 and the shaded bars the CDCactivities of KM966.

FIG. 23 graphically shows the CDC activities of KM966 against the humanneuroblastoma cell line GOTO and human brain tumor cell line A172. Theordinate indicates the cytotoxic activity and the abscissa theconcentration of the antibody added. The solid bars indicate the CDCactivities of KM696 and the shaded bars the CDC activities of KM966.

FIG. 24 graphically shows the ADCC (antibody dependent cell mediatedcytotoxicity) activities of KM966 against the human lung small cellcarcinoma cell lines SBC-3 and LU-135. The ordinate indicates thecytotoxic activity and the abscissa the concentration of the antibodyadded. The solid bars indicate the ADCC activities of KM-696 and theshaded bars the ADCC activities of KM966.

FIG. 25 graphically shows the ADCC activities of KM966 against the humanlung squamous carcinoma cell line PC-10 and human lung adenocarcinomacell line RERF-LC-MS. The ordinate indicates the cytotoxic activity andthe abscissa the concentration of the antibody added. The solid barsindicate the ADCC activities of KM-696 and the shaded bars the ADCCactivities of KM966.

FIG. 26 graphically shows the ADCC activities of KM966 against the humanlung large cell carcinoma cell line PC-13 and human neuroblastoma cellline NAGAI. The ordinate indicates the cytotoxicity and the abscissa theconcentration of the antibody added. The solid bars indicate the ADCCactivities of KM-696 and the shaded bars the ADCC activities of KM966.

FIG. 27 graphically shows the ADCC activities of KM966 against the humanneuroblastoma cell line GOTO and human brain tumor cell line A172. Theordinate indicates the cytotoxicity and the abscissa the concentrationof the antibody added. The solid bars indicate the ADCC activities ofKM-696 and the shaded bars the ADCC activities of KM966.

FIG. 28 shows a restriction enzyme cleavage map of a 9.3 kb XbaIfragment of the KM50 cell chromosomal DNA.

FIG. 29 shows a construction scheme for a plasmid, pKMB11.

FIG. 30 shows a construction scheme for a plasmid, pKMD6.

FIG. 31 shows a construction scheme for a plasmid, pEPKMA1.

FIG. 32 shows a construction scheme for a plasmid, pEPKMB1.

FIG. 33 shows a construction scheme for a plasmid, pAGE501.

FIG. 34 shows a construction scheme for a plasmid, pAGE109.

FIG. 35 shows a construction scheme for a plasmid, pAGE502.

FIG. 36 shows a construction scheme for a plasmid, pAGE503.

FIG. 37 shows a construction scheme for a plasmid, pSEd1.

FIG. 38 shows a construction scheme for a plasmid, pSE1D2.

FIG. 39 shows a construction scheme for a plasmid, pIG1SE1d2.

FIG. 40 shows a construction scheme for a plasmid, pIG1SE1d3.

FIG. 41 shows a construction scheme for a plasmid, pIG1SE1d4.

FIG. 42 shows a construction scheme for a plasmid, pPMOL2.

FIG. 43 shows a construction scheme for a plasmid, pPMOL3.

FIG. 44 shows a construction scheme for a plasmid, pchCKA7.

FIG. 45 shows a construction scheme for a plasmid, pchCKB1.

FIG. 46 shows a construction scheme for a plasmid, pckCKC1.

FIG. 47 shows a construction scheme for a plasmid, pChiIgHB2.

FIG. 48 shows a construction scheme for a plasmid, pChiIgLA1.

FIG. 49 shows a construction scheme for plasmids, pKM641HA3 andpKM641LA2.

FIG. 50 shows a construction scheme for a plasmid, pChi641HA1.

FIG. 51 shows a construction scheme for a plasmid, pKM641HE1.

FIG. 52 shows a construction scheme for a plasmid, pKM641HF1.

FIG. 53 shows a construction scheme for a plasmid, pChi641HA1.

FIG. 54 shows a construction scheme for a plasmid, pChi641HAM1.

FIG. 55 shows a construction scheme for a DNA, hKM796H.

FIG. 56 shows a construction scheme for a DNA, hKM796L.

FIG. 57 shows a construction scheme for a plasmid, phKM796HM1.

FIG. 58 shows a construction scheme for a plasmid, phKM796HMS1.

FIG. 59 shows a construction scheme for a plasmid, phKM796LI1.

FIG. 60 shows a construction scheme for a plasmid, phKM796LM1.

FIG. 61 shows a construction scheme for a plasmid, phKM796LMS1.

FIG. 62 shows a construction scheme for a plasmid, phKM796H107.

FIG. 63 shows a construction scheme for a plasmid, phKM796HL1.

FIG. 64 shows a construction scheme for a plasmid named pBSA.

FIG. 65 shows a construction scheme for a plasmid named pBSAE.

FIG. 66 shows a construction scheme for a plasmid named pBSH-S.

FIG. 67 shows a construction scheme for a plasmid named pBSK-H.

FIG. 68 shows a construction scheme for plasmids named PBSH-SA andpBSK-HA.

FIG. 69 shows a construction scheme for plasmids named pBSH-SAE andpBSK-HAE.

FIG. 70 shows a construction scheme for plasmids named PBSH-SAEE andpBSK-HAEE.

FIG. 71 shows a construction scheme for a plasmid named pBSK-HAEESal.

FIG. 72 shows a construction scheme for a plasmid named pBSX-S.

FIG. 73 shows a construction scheme for a plasmid named pBSX-SA.

FIG. 74 shows a construction scheme for a plasmid named pBSSC.

FIG. 75 shows a construction scheme for a plasmid named pBSMo.

FIG. 76 shows a construction scheme for a plasmid named pBSMoS.

FIG. 77 shows a construction scheme for a plasmid named pChiIgLA1S.

FIG. 78 shows a construction scheme for a plasmid named pMohCK.

FIG. 79 shows a construction scheme for a plasmid named pBSMoSal.

FIG. 80 shows a construction scheme for a plasmid named pBSMoSalS.

FIG. 81 shows a construction scheme for a plasmid named pBShCγ1.

FIG. 82 shows a construction scheme for a plasmid named pMohCγ1.

FIG. 83 shows a construction scheme for a plasmid named pMoγ1SP.

FIG. 84 shows a construction scheme for a plasmid named pMOKγ1SP.

FIG. 85 shows a construction scheme for a plasmid named pKANTEX93.

FIG. 86 shows a construction scheme for a plasmid named pBSNA.

FIG. 87 shows a construction scheme for a plasmid named pBSH3.

FIG. 88 shows a construction scheme for a plasmid named pBSES.

FIG. 89 shows a construction scheme for a plasmid named pBSL3.

FIG. 90 shows a construction scheme for a plasmid named pKANTEX796H.

FIG. 91 shows a construction scheme for a plasmid named pKANTEX796.

FIG. 92 shows a construction scheme for a plasmid named pT796.

FIG. 93 is a graphic representation of transient human anti-GM₂ chimeraantibody expression by the plasmids pKANTEX796 and pT796. The ordinatedonotes the antibody concentration that showed GM₂ -binding activity,and the abscissa denotes the time after introduction of the plasmid.

FIG. 94 shows a construction scheme for a plasmid named pESH10.

FIG. 95 shows a construction scheme for a plasmid named pBSL16.

FIG. 96 illustrates a process for mutagenesis by PCR and a process forcloning DNA fragments mutated.

FIG. 97 shows a construction scheme for a plasmid named pBSLV1+2.

FIG. 98 shows a construction scheme for a plasmid named pBSLm-28.

FIG. 99 shows a construction scheme for a plasmid named pBSHSGL.

FIG. 100 shows a construction scheme for a plasmid named pT796LCDR.

FIG. 101 shows a construction scheme for plasmids named pT796HLCDR,pT796HLCDRHV2 and pT796HLCDRHV4.

FIG. 102 shows a construction scheme for a plasmid named pT796HLCDRH10.

FIG. 103 shows construction scheme for plasmids named pT796HCDR,pT796HCDRHV2, pT796HCDRHV4 and pT796HCDRH10.

FIG. 104 is a graphic representation of the results of human CDR-graftedanti-GM₂ antibody activity evaluation in terms of transient expressionas obtained using the plasmids pT796, pT796HCDR, pT796HCDRHV2,pT796HCDRHV4 and pT796HCDRH10. The ordinate denotes the plasmid used,and the abscissa denotes the relative activity value with the activityobtained with the chimera antibody being taken as 100%.

FIG. 105 shows a construction scheme for plasmids named pT796HLCDRLV1,pT796HLCDRLV2, pT796HLCDRLV3, pT796HLCDRLV4, pT796HLCDRLV8,pT796HLCDRLm-2, pT796HLCDRLm-8, pT796HLCDRLm-28 and pT796HLCDRHSGL.

FIG. 106 is a graphic representation of the results of human CDR-graftedanti-GM₂ antibody activity evaluation in terms of transient expressionas obtained using the plasmids pT796, pT796HLCDR, pT796HLCDRLV1,pT796HLCDRLV2, pT796HLCDRLV3, pT796HLCDRLV4, pT796HLCDRLV8,pT796HLCDRLm-2, pT796HLCDRLm-8, pT796HLCDRLm-28 and pT796HLCDRHSGL. Theordinate denotes the plasmid used, and the abscissa denotes the relativeactivity value with the activity obtained with the chimera antibodybeing taken as 100%.

FIG. 107 shows a construction scheme for plasmids namedpKANTEX796HLCDRLm-28 and pKANTEX796HLCDRHSGL.

FIG. 108 shows electrophoretic patterns obtained for human anti-GM₂chimera antibody KM966 and purified human CDR-grafted anti-GM₂antibodies KM8966 and KM8967 by SDS-PAGE (4 to 15% gradient gels used).The patterns shown on the left side are those obtained under reducingconditions, and those on the right under nonreducing conditions. Fromthe left of each lane, the electrophoretic patterns forhigh-molecular-weight marker, KM966, KM8966, KM8967,low-molecular-weight marker, KM966, KM8966 and KM8967 are shown in thatorder.

FIG. 109 is a graphic representation of the GM₂ -binding activities ofhuman anti-GM₂ chimera antibody KM966 and purified human CDR-graftedanti-GM₂ antibodies KM8966 and KM8967. The ordinate denotes the GM₂-binding activity, and the abscissa the antibody concentration. Theplots ∘,  and Δ stand for the GM₂ -binding activities of KM966, KM8966and KM8967, respectively.

FIG. 110 is a graphic representation of the reactivities of humananti-GM₂ chimera antibody KM966 and purified human CDR-grafted anti-GM₂antibodies KM8966 and KM8967 against various gangliosides. The ordinatedenotes the ganglioside species, and the abscissa the binding activity.AcGM2 stands for N-acetyl-GM₂, GcGM2 for N-glycolyl-GM₂, AcGM3 forN-acetyl-GM₃ and GcGM3 for N-glycolyl-GM₃. The plots □, ▪ and □ standfor the reactivities of KM966, KM8966 and KM8967, respectively.

FIG. 111 is a graphic representation of the reactivities of humananti-GM₂ chimera antibody KM966 and purified human CDR-grafted anti-GM₂antibodies KM8966 and KM8967 against the human lung small cell carcinomacell line SBC-3. The ordinate denotes the number of cells, and theabscissa the fluorescence intensity. From the lowermost graph, thereactivities of control, KM8967, KM8966 and KM966 are shown in thatorder.

FIG. 112 graphically shows the CDC activities of chimeric human anti-GM₂antibody KM966 and purified human CDR-grafted anti-GM₂ antibodies KM8966and KM8967 against the human lung small cell carcinoma cell line SBC-3.The ordinate indicates the cytotoxic activity and the abscissa theconcentration of the antibody. The plots □, ▪ and □ stand for the CDCactivities of KM966, KM8966 and KM8967, respectively.

FIG. 113 graphically shows the ADCC activities of chimeric humananti-GM₂ antibody KM966 and purified human CDR-grafted anti-GM₂antibodies KM8966 and KM8967 against the human lung small cell carcinomacell line SBC-3. The ordinate indicates the cytotoxicity and theabscissa the concentration of the antibody. The plots □, ▪ and □ standfor the ADCC activities of KM966, KM8966 and KM8967, respectively.

FIG. 114 shows a construction scheme for plasmids, pKANTEX796HM1Lm-28,pKANTEX796HM2Lm-28, pKANTEX796HM3Lm-28, pKANTEX796HM31Lm-28 andpKANTEX796HM32Lm-28.

FIG. 115 shows the electrophoretic patterns in SDS-PAGE (using 4-15%gradient gels) of chimeric human anti-GM₂ antibody KM966, humanCDR-grafted anti-GM₂ antibody KM8966 and human CDR-grafted anti-GM₂antibodies each having different mutation. The pattern obtained undernonreducing conditions is shown on the left side and that obtained underreducing conditions on the right side. M stands for molecular weightmarkers (from the top, the arrows indicate the molecular weight of 205Kd, 140 Kd, 83 Kd, 45 Kd, 32.6 Kd, 18 Kd and 7.5 Kd in that order) and1, 2, 3, 4, 5, 6 and 7 stand for the electrophoretic patterns of KM966,KM8966, M1-28, M2-28, M3-28, M31-28 and M32-28, respectively.

FIG. 116 graphically shows the CDC activities of chimeric human anti-GM₂antibody KM966, human CDR-grafted anti-GM₂ antibody KM8966 and humanCDR-grafted anti-GM₂ antibodies each having different mutation againstthe human lung small cell carcinoma cell line SBC-3. The ordinateindicates the cytotoxic activity and the abscissa the concentration ofthe antibody. The plots □, ▪, □, □, □, □ and □ stand for the CDCactivities of KM966, KM8966, M1-28, M2-28, M3-28, M31-28 and M32-28,respectively.

FIG. 117 shows a construction scheme for plasmids, pKANTEX796HLm-28No.1, pKANTEX796HM1Lm-28 No.1, pKANTEX796HM2Lm-28 No.1 andpKANTEX796HM3Lm-28 No.1.

FIG. 118 shows the electrophoretic patterns in SDS-PAGE (using 4-15%gradient gels) of chimeric human anti-GM₂ antibody KM966 and humanCDR-grafted anti-GM₂ antibodies each having different mutation. Thepattern obtained under nonreducing conditions is shown on the left sideand that obtained under reducing conditions on the right side. M standsfor molecular weight markers (from the top, the arrows indicate themolecular weight of 205 Kd, 140 Kd, 83 Kd, 45 Kd, 32.6 Kd, 18 Kd and 7.5Kd in that order) and 1, 2, 3, 4 and 5 stand for the electrophoreticpatterns of KM966, h796H-No.1, M1-No.1, M2-No.1 and M3-No.1,respectively.

FIG. 119 graphically shows the CDC activities of chimeric human anti-GM₂antibody KM966, human CDR-grafted anti-GM₂ antibodies KM8966 and KM8970and human CDR-grafted anti-GM₂ antibodies each having different mutationagainst the human lung small cell carcinoma cell line SBC-3. Theordinate indicates the cytotoxic activity and the abscissa theconcentration of the antibody. The plots □, ▪, □, □, □, □ and □ standfor the CDC activities of KM966, KM8966, KM8970, h796H-No.1, M1-No.1,M2-No.1 and M3-No.1, respectively.

FIG. 120 graphically shows the GM₂ -binding activities of chimeric humananti-GM₂ antibody KM966 and human CDR-grafted anti-GM₂ antibodies KM8969and KM8970. The ordinate indicates the GM₂ -binding activity and theabscissa the concentration of the antibody. The plots ∘,  and ▴ standfor the GM₂ -binding activities of KM966, KM8969 and KM8970,respectively.

FIG. 121 graphically shows the reactivities of chimeric human anti-GM₂antibody KM966 and human CDR-grafted anti-GM₂ antibodies KM8969 andKM8970 against various gangliosides. The ordinate indicates theganglioside species and the abscissa the binding activity. AcGM₂ standsfor N-acetyl-GM₂, GcGM₂ for N-glycolyl-GM₂, AcGM₃ for N-acetyl-GM₃ andGcGM₃ for N-glycolyl-GM₃. The plots □, ▪ and □ stand for the GM₂-binding activities of KM966, KM8969 and KM8970, respectively.

FIG. 122 graphically shows the reactivities of chimeric human anti-GM₂antibody KM966 and human CDR-grafted anti-GM₂ antibodies KM8969 andKM8970 against the human lung small cell carcinoma cell line SBC-3. Theordinate indicates the number of cells and the abscissa the fluorescenceintensity. From the lowermost graph, the reactivities of control, KM966,KM8970 and KM8969 are shown in that order.

FIG. 123 graphically shows the ADCC activities of chimeric humananti-GM₂ antibody KM966 and human CDR-grafted anti-GM₂ antibodies KM8966, KM8969 and KM8970 against the human lung small cell carcinoma cellline SBC-3. The ordinate indicates the cytotoxicity and the abscissa theconcentration of the antibody. The plots □, ▪, □ and □ stand for theADCC activities of KM966, KM8966, KM8969 and KM8970, respectively.

FIG. 124 graphically shows the CDC activities of chimeric human anti-GM₂antibody KM966 and human CDR-grafted anti-GM₂ antibodies KM 8966, KM8969and KM8970 against the human lung small cell carcinoma cell line SBC-3obtained when the reaction was carried out for 1 hour and 4 hours afterthe addition of the human complement. The ordinate indicates thecytotoxicity and the abscissa the concentration of the antibody. Theplots □, ▪, □, □, □, □, □ and □ stand for the CDC activities of KM966 (1hour), KM966 (4 hours), KM8966 (1 hour), KM8966 (4 hours), KM8969 (1hour), KM8969 (4 hours), KM8970 (1 hour) and KM8970 (4 hours).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to humanized antibodies specific for theganglioside GM₂. The antibodies can be of any of the immunoglobulin (Ig)classes, it is preferable, however, that the antibodies be of the IgGtype. The term "humanized antibody", as used herein, includes within itsmeaning, chimeric human antibodies and CDR-grafted antibodies. Chimerichuman antibodies of the invention include the V_(H) and V_(L) of anantibody of an animal other than a human and the C_(H) and C_(L) of ahuman antibody. The CDR-transplanted antibodies of the invention resultfrom the replacement of CDRs of the V_(H) and V_(L) of a human antibodywith those of the V_(H) and V_(L), respectively, of an antibody of ananimal other than a human.

An example of a chimeric human antibody of the invention is an antibodythe V_(H) of which contains an amino acid sequence segment as defined bySEQ ID NO:1, including the 1st to 120th amino acids of that sequence,and the V_(L) of which contains an amino acid sequence segment asdefined by SEQ ID NO:2, including the 1st to 107th amino acids of thatsequence.

An example of a CDR-grafted antibody of the invention is an antibody theV_(H) CDRs of which have the amino acid sequences defined by SEQ IDNO:6, SEQ ID NO:7 and SEQ ID NO:8 and the V_(L) CDRs of which have theamino acid sequences defined by SEQ ID NO:9, SEQ ID NO:10 and SEQ IDNO:11.

The chimeric human antibodies of the invention can be produced in thefollowing manner:

(1) Preparation of cDNAs Coding for the V_(H) and V_(L) of an Antibodyof Nonhuman Animal

cDNAs coding for the V_(H) and V_(L) of an antibody of a nonhumananimal, for example a mouse anti-GM₂ monoclonal antibody, can beprepared as follows.

mRNAs can be extracted from hybridomas producing the mouse anti-GM₂monoclonal antibody, for example hybridomas producing the mouse anti-GM₂monoclonal antibody KM796, and cDNAs reverse transcribed therefrom.Using the cDNAs, a library can be constructed using phage or plasmidvectors. The recombinant phage or recombinant plasmid containing thecDNA coding for the V_(H), and the recombinant phage or recombinantplasmid containing the cDNA coding for the V_(L) can be isolated fromthe library using a constant region portion or a variable region portionof an antibody of a nonhuman animal, for example a mouse antibody, as aprobe. The base sequences of the V_(H) -encoding cDNA and V_(L)-encoding CDNA in the recombinant phage or recombinant plasmid can thenbe determined. Examples of the nonhuman animals include mice, rats,hamsters and monkeys.

(2) Construction of a Vector for Chimeric Human Antibody Expression

Expression of chimeric human antibody H chain and L chains can beeffected using expression vectors suitable for use in animal cells,inserted into which are the cDNAs coding for the human C_(H) and C_(L).Any expression vector suitable for use in animal cells can be used,provided that it allows integration and expression of the human antibodyconstant region-encoding cDNAs. Examples include pAGE107[Cytotechnology, 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307(1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci.U.S.A., 78, 1527 (1981)] and pSG1βd2-4 [Cytotechnology, 4, 173 (1990)],among others. Examples of promoters and enhancers suitable for use insuch expression vectors include the SV40 early promoter and enhancer [J.Biochem., 101, 1307 (1987)], the Moloney mouse leukemia virus LTR (longterminal repeat) promoter and enhancer [Biochem. Biophys. Res. Commun.,149, 960 (1987)] and the immunoglobulin H chain promoter [Cell, 41, 479(1985)] and enhancer [Cell, 33, 717 (1983)]. The promoters and enhancersare located in the expression vector in operable linkage with the codingsequences.

(3) Construction of a Chimeric Human Antibody Expression Vector

The vector for chimeric human antibody H chain and L chain expression,as obtained in (2), is provided with a cloning site upstream of thehuman constant region, for insertion of a cDNA coding for the variableregion of an antibody of a nonhuman animal. Insertion, at this cloningsite, of the cDNA coding for the variable region of a nonhuman animalantibody, using a synthetic DNA comprising a 5' terminal base sequenceof the human antibody constant region and a 3' terminal base sequence ofthe variable region of the nonhuman animal antibody and havingrestriction enzyme sites on both ends, gives a chimeric human antibodyexpression vector with the cDNA coding for the human antibody constantregion and the cDNA coding for the variable region of the nonhumananimal antibody joinedly inserted therein via the synthetic DNA forproducing appropriate restriction enzyme sites. The synthetic DNA can besynthesized using a DNA synthesizer based on the 5' terminal basesequence of the human antibody constant region and the base sequence ofsaid 3' terminal base sequence of the nonhuman animal antibody variableregion.

(4) Construction of a Chimeric Human Antibody H Chain Expression Vector

A vector for chimeric human antibody H chain expression is constructed,for example, by excising that portion of the human antibody C_(H)-encoding cDNA which covers from the ApaI site near the 5' terminus tothe 3' terminus and inserting that portion into an expression vectorsuitable for use in animal cells. This vector for chimeric humanantibody H chain expression is provided with a cloning site forinsertion of a CDNA coding for a nonhuman animal V_(H). CDNA coding forthe nonhuman animal V_(H), excised using an appropriate restrictionenzyme, is inserted into the vector at the cloning site using asynthetic DNA comprising that portion of the human antibody C_(H) genewhich covers from the 5' terminus to the ApaI site and the base sequenceof a 3' terminal portion of the nonhuman animal antibody V_(H) gene andhaving restriction enzyme sites on both ends, to give a chimeric humanantibody H chain expression vector which allows no change in the aminoacid sequence of V_(H) upon expression thereof and has appropriaterestriction enzyme sites.

(5) Construction of a Chimeric Human Antibody L Chain Expression Vector

A vector for chimeric human antibody L chain expression is constructed,for example by introducing an EcoRV site into the human antibody C_(L)-encoding CDNA in the vicinity of the 5' terminus by mutagenesis,excising that portion which covers from the EcoRV site to the 3'terminus and inserting that portion into a plasmid, such as the plasmidpIg1SE1d4. This vector for chimeric human antibody L chain expression isprovided with a cloning site for insertion of the CDNA coding fornonhuman animal V_(L). The nonhuman animal antibody V_(L) -encodingCDNA, excised with an appropriate restriction enzyme, is inserted intothe vector at the cloning site using a synthetic DNA comprising thatportion of the human antibody C_(L) gene which covers from the 5'terminus to the EcoRV site and the base sequence of a 3' terminalportion of the nonhuman animal antibody V_(L) gene and havingrestriction enzyme sites on both ends, to give a chimeric human antibodyL chain expression vector which allows no change in the amino acidsequence of V_(L) upon expression thereof.

(6) Introduction of the Chimeric Human Antibody Expression Vectors IntoHost Cells

Introduction of the chimeric human antibody H chain expression vectorand chimeric human antibody L chain expression vector into host cellsgives a transformant producing the chimeric human antibody. Inintroducing the vectors into host cells, a splicing signal may beintroduced into the chimeric human antibody H chain and L chainexpression vectors for mRNA stabilization [Cell, 17, 737 (1979)].

The chimeric human antibody H chain and L chain vectors can beintroduced into host cells, for example, simultaneously byelectroporation [JP-A-2-257891 (the term "JP-A" used herein means anunexamined published Japanese patent application.); Cytotechnology, 3,133 (1990)]. In addition, an expression vector containing genes codingfor both the chimeric human antibody H chain and L chain [tandemexpression vector] can be introduced into host cells [BIO/TECHNOLOGY, 9,64 (1991)]. The use of a tandem expression vector is preferred since ahigher level of chimeric human antibody expression can be attainedthereby, with approximately equal H chain and L chain expression levels.

An example of a method of producing the CDR-grafted antibodies of theinvention is described as follows.

First, a CDR-grafted antibody expression vector can be constructed bythe method of Winter et al. [Nature, 332, 323 (1988)] as follows.

Three synthetic DNAs are constructed designed so as to comprise thecDNAs coding for three CDR peptides of the V_(H) of a nonhuman animalantibody, for example, peptides having the amino acid sequences definedby SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, with DNAs coding for aminoacid sequences comprising of several amino acids from both ends of thecorresponding CDRs of the V_(H) of a human antibody being located at therespective both ends of the cDNAs, DNA synthesis is carried out with aplasmid containing the human antibody V_(H) gene as a template. Anexample of the human antibody V_(H) gene-containing plasmid is the M13plasmid containing a human antibody NEW gene-derived sequence [J. Biol.Chem., 253, 585 (1978); Nature, 332, 323 (1988)].

The DNA obtained is inserted into the vector for chimeric human antibodyH chain expression in the same manner as in the construction of thechimeric human antibody expression vector mentioned above to give aCDR-grafted antibody H chain expression vector.

Similarly, using, as primers, three synthetic DNAs designed to comprisethe cDNAs coding for three CDR peptides of the V_(L) of a nonhumananimal antibody, for example, the peptides having the amino acidsequences defined by SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, withDNAs coding for amino acid sequences comprising several amino acids fromboth ends of the corresponding CDRs of the human antibody V_(L) beinglocated at the respective both ends of said cDNAs, DNA synthesis iscarried out with a human antibody V_(L) gene-containing plasmid as atemplate. An example of the human antibody V_(L) gene-containing plasmidis the M13 plasmid containing a human myeloma protein (Bence-Jonesprotein) REI gene-derived sequence [Eur. J. Biochem., 45, 513 (1974);Nature, 332, 323 (1988)].

By inserting the DNA obtained into a vector for chimeric human L chainexpression in the same manner as described in respect of theconstruction of the chimeric human antibody expression vector, aCDR-grafted antibody L chain expression vector can be constructed.

It is also possible to construct the CDR-grafted antibody H chain and Lchain expression vectors by synthesizing DNAs coding for the peptideshaving amino acid sequences resulting from replacement of the three CDRseach of the H chain and L chain of a human antibody with thecorresponding CDRs of the H chain and L chain of a nonhuman animalantibody and then inserting the DNAs into a vector for chimeric humanantibody H chain or L chain expression in the same manner as describedin respect of the construction of the chimeric human antibody expressionvector mentioned above.

The CDR-grafted antibody expression vector can be introduced into hostcells in the same manner as the chimeric human antibody expressionvector to give a transformant producing the CDR-grafted antibody.

The host cells suited for the introduction there into of the chimerichuman antibody or CDR-grafted antibody expression vector may be any hostcells provided that the chimeric human antibody or CDR-grafted antibodycan be expressed therein. Examples include mouse SP2/0-Ag14 cells (ATCCCRL1581; hereinafter, "SP2/0 cells"), mouse P3X63-Ag8.653 cells (ATCCCRL1580), CHO cells deficient in the dihydrofolate reductase gene(hereinafter, "dhfr") [Urlaub et al.: Proc. Natl. Acad. Sci. U.S.A., 77,4216 (1980)] and rat YB2/3HL.P2.G11.16Ag.20 cells (ATCC CRL1662;hereinafter, "YB2/0 cells"), with YB2/0 cells being preferred.

The transformants producing the chimeric human antibody or CDR-graftedantibody are selected by the method disclosed in JP-A-2-257891 usingPRMI1640 medium containing G418 and fetal calf serum. A particularexample of the chimeric human antibody-producing transformant is thetransformant KM966 producing a chimeric human antibody that reacts withthe ganglioside GM₂. Examples of human CDR-grafted antibody-producingtransformants include the transformants KM8966 and KM8967 each producinga human CDR-grafted antibody that reacts with the ganglioside GM₂. KM966has been deposited with the Fermentation Research Institute, Agency ofIndustrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki 305 JAPAN, as of Jul. 15, 1992 under the deposit number FERMBP-3931. KM8966 and KM8967 have also deposited with the above-describedinstitute as of May 23, 1995 under the deposit numbers FERM BP-5105 andFERM BP-5106, respectively.

When the transformant obtained is cultivated in a medium, the chimerichuman antibody or CDR-grafted antibody can be produced and accumulatedin the culture fluid. The activity of the chimeric human antibody orCDR-grafted antibody in the medium can be determined by an enzyme-linkedimmunosorbent assay (ELISA; E. Harlow et al. (ed.): Antibodies--ALaboratory Manual, Cold Spring Harbor Laboratory, 1988). The antibodyproductivity of the transformant can be increased by utilizing a dhframplification system as disclosed in JP-A-2-257891.

The chimeric human antibody and CDR-grafted antibody can be purifiedfrom the culture supernatants obtained as mentioned above using aprotein A column (E. Harlow et al. (ed.): Antibodies--A LaboratoryManual, Cold Spring Harbor Laboratory, 1988). As noted above, thechimeric human antibody KM966, which reacts with the ganglioside GM₂, isa specific example of the thus-obtained chimeric human antibodies andCDR-grafted antibodies.

The reactivity of the chimeric human antibody or CDR-grafted antibody ofthe invention can be checked by ELISA. The molecular weight of thepurified antibody H chain or L chain or whole antibody molecule can bedetermined by polyacrylamide gel electrophoresis (SDS-PAGE) or Westernblotting (E. Harlow et al. (ed.): Antibodies--A Laboratory Manual, ColdSpring Harbor Laboratory, 1988).

The binding activity of the chimeric human antibody or CDR-graftedantibody that reacts with the ganglioside GM₂ of cultured cancer cellscan be measured, for example, by the fluorescent antibody technique orby ELISA. The complement dependent cytotoxic activity (CDC activity) andantibody dependent cell mediated cytotoxic activity (ADCC activity) ofthe chimeric human antibody or CDR-grafted antibody are measured by themethods of Ohta et al. [Cancer Immunol. Immunother., 36, 260 (1993)].

The humanized antibodies of the invention specifically bind to humancancer cells and exhibit CDC activity and ADCC activity against humancancer cells and therefore are useful in the treatment of human cancers,among others.

The humanized antibodies according to the present invention can be usedalone as an anticancer agent. They may be formulated into an anticancercomposition together with at least one pharmaceutically acceptablecarrier. For instance, the humanized antibodies are dissolved inphysiological saline, an aqueous solution of glucose, lactose ormannitol and the like. The powder of the humanized antibodies forinjection can be prepared by lyophilizing the humanized antibodies inaccordance with the conventional method and mixing the lyophilizedproducts with sodium chloride. The anticancer composition may furthercontain additives conventionally used well known in the art of medicalpreparation, for example, pharmaceutically acceptable salts.

The humanized antibodies according to the present invention can beadministered in the form of the above-described anticancer compositionto mammals including human in a dose of 0.2 to 20 mg/kg/day. The dosemay vary depending on the age, condition, etc. of patients. Theadministration of the anticancer composition can be effected byintravenous injection once a day (single administration or consecutiveadministration) or intermittently one to three times a week or onceevery two to three weeks.

The anticancer composition is expected to be useful for treating cancersuch as melanoma, neuroblastoma and glioma.

The following Examples and Reference Examples are further illustrativeof the present invention, but are not to be construed to limit the scopeof the present invention.

EXAMPLE 1 Production of Chimeric Human Anti-GM₂ Antibodies

1. Isolation of mRNAs from Hybridoma Cells Producing the Mouse Anti-GM₂Monoclonal Antibody KM-796 or KM-750 and from Hybridoma Cells Producingthe Rat Anti-GM₂ Monoclonal Antibody KM-603

Using mRNA extraction kit Fast Track (product number K1593-02)manufactured by Invitorogen and following the description of the manualattached to the kit, mRNAs were isolated from 1×10⁸ cells each of themouse anti-GM₂ monoclonal antibody KM-796-producing hybridoma cell line(FERM BP-3340), the mouse anti-GM₂ monoclonal antibody KM-750-producinghybridoma cell line (FERM BP-3339) and the rat anti-GM₂ monoclonalantibody KM-603-producing hybridoma cell line (FERM BP-2636).

2. Construction of Monoclonal Antibody KM-796 and KM-750 H Chain and LChain cDNA Libraries

Using cDNA Synthesis Kit (product number 27-9260-01) manufactured byPharmacia and following the manual attached to the kit, cDNA having theEcoRI adapter on both ends was synthesized from 5 μg each of the KM-796-and KM-750-derived mRNAs obtained as described in Paragraph 1 above.About 6 μg of each cDNA product obtained was dissolved in 10 μl ofsterilized water and fractionated by agarose gel electrophoresis, and acDNA fragment (about 1.8 kb) corresponding to the IgG antibody H chainand a cDNA fragment (about 1.0 kb) corresponding to the L chain wererecovered (about 0.1 μg each). Then, 0.1 μg of each cDNA fragment ofabout 1.8 kb and 0.1 μg of each cDNA fragment of about 1.0 kb wererespectively dissolved in 11.5 μl of T4 ligase buffer, together with 1μg of the Lambda ZAPII vector (cleaved with EcoRI and then treated withcalf intestine alkaline phosphatase; product of Stratagene). Afteraddition of 175 units of T4 DNA ligase, each solution was incubated at12° C. for 24 hours and then at room temperature for 2 hours. A 4-μlportion of each reaction mixture was subjected to packaging into thelambda phage in the conventional manner [Maniatis et al. (ed.):Molecular Cloning, 2.95 Cold pring Harbor Laboratory, 1989] using GigaPak Gold (Stratagene), followed by transfection, in the conventionalmanner [Maniatis et al. (ed.): Molecular Cloning, 2.95-107, Cold SpringHarbor Laboratory, 1989] of the Escherichia coli strain XL1-Blue[Biotechniques, 5, 376 (1987)] attached to Giga Pak Gold, to give about4×10³ phage clones each as a KM-796 or KM-750 H chain or L chain cDNAlibrary. Then the phage clones of each library were fixed on anitrocellulose filter in the conventional manner [Maniatis et al. (ed.):Molecular Cloning, 2.112, Cold Spring Harbor Laboratory, 1989].

3. Construction of KM-603 H Chain and L Chain cDNA Libraries

Using 5 μg of the KM-603 mRNA obtained as mentioned above in Paragraph 1and cDNA Synthesis Kit (product number 27-9260-01) manufactured byPharmacia, cDNA having the EcoRI adapter on both ends was synthesized.About 6 μg of the cDNA produced was dissolved in 10 μl of sterilizedwater and fractionated by agarose gel electrophoresis. A cDNA fragment(about 2.2 kb) corresponding to the IgG antibody H chain and a cDNAfragment (about 1.0 kb) corresponding to the L chain were recovered(about 0.1 μg each). Then 0.1 μg of the cDNA fragment of about 2.2 kband 0.1 μg of the cDNA fragment of about 1.0 kb were respectivelydissolved in 11.5 μl of T4 ligase buffer, together with 1 μg of theLambda ZAPII vector (cleaved with EcoRI and then treated with calfintestine alkaline phosphatase; product of Stratagene) and, afteraddition of 175 units of T4 DNA ligase, the resultant solution wasincubated at 12° C. for 24 hours and then at room temperature for 2hours. A 4-μl portion of each reaction mixture was subjected topackaging into the lambda phage in the conventional manner [Maniatis etal. (ed.): Molecular Cloning, 2.95, Cold Spring Harbor Laboratory, 1989]using Giag Pak Gold (Stratagene), followed by transfection, in theconventional manner [Maniatis et al. (ed.): Molecular Cloning, 2.95-107,Cold Spring Harbor Laboratory, 1989], of the Escherichia coli strainXL-Blue attached to Giga Pak Gold, whereby about 1×10⁴ phage clones wereobtained each as a KM-603 H chain or L chain cDNA library. Then, thephage clones of each library were fixed on a nitrocellulose filter inthe conventional manner [Maniatis et al. (ed.): Molecular Cloning,2.112, Cold Spring Harbor Laboratory, 1989].

4. Cloning of the KM-796 and KM-750 H Chain and L Chain cDNAs

From among the KM-796 and KM-750 H chain cDNA libraries and L chain cDNAlibraries constructed as described above in Paragraph 2, phage clonesfirmly bound at 65° C. to a probe prepared by labeling a mouseimmunoglobulin constant region cDNA [for the H chain, the BamHI-XhoIfragment of the mouse Cγ3 cDNA (Wels et al: EMBO J., 3, 2041-2046,1984); for the L chain, the HpaI-XhoI fragment of the mouse CK cDNA(Hieter et at.: Cell, 22, 197-207, 1980)] with ³² P were recovered inthe conventional manner [Maniatis et al.: Molecular Cloning, 2.108, ColdSpring Harbor Laboratory, 1989]. Then, using a ZAP-cDNA Synthesis Kit(cDNA synthesis kit; product number sc200400) manufactured byStratagene, phage clones were converted into pBluescript plasmids, and aKM-796 H chain cDNA-containing recombinant plasmid (pKM796H1) and aKM-796 L chain cDNA-containing recombinant plasmid (pKM796L1) (FIG. 1)as well as a KM-750 H chain cDNA-containing recombinant plasmid(pKM750H1) and a KM-750 L chain cDNA-containing recombinant plasmid(pKM750L1) (FIG. 2) were obtained. Cleavage of pKM796H1, pKM750H1,pKM796L1 and pKM750L1 with EcoRI revealed that a cDNA fragment of about1.8 kb had been inserted into pKM796H1 and pKM750H1 and a cDNA fragmentof about 0.9 kb into pKM796L1 and pKM750L1.

5. Cloning of KM-603 H Chain and L Chain cDNAs

Phage clones firmly bound at 65° C. to a probe prepared by labeling amouse immunoglobulin constant region chromosomal gene [mouse Cμgene-containing SmaI-KpnI fragment of about 11.5 kb (Kataoka et al.:Proc. Natl. Acad. Sci. U.S.A., 77, 919-923, 1980) and mouse CKgene-containing HindIII-BamHI fragment of about 3 kb (Sakano et al.:Nature, 280, 288, 1979)] with ³² P were isolated from the KM-603 H chaincDNA library and L chain cDNA library constructed as mentioned above inParagraph 3 in the conventional manner [Maniatis et al. (ed.): MolecularCloning, 2.108, Cold Spring Harbor Laboratory, 1989]. Then, usingZAP-cDNA Synthesis kit (product number sc200400) manufactured byStratagene, the phage clones were converted to pBluescript plasmids anda KM-603 H chain cDNA-containing recombinant plasmid, pKM603H1, and aKM-603 L chain cDNA-containing recombinant plasmid, pKM603L1, wereobtained (FIG. 3). Cleavage of pKM603H1 and pKM603L1 revealed thatpKM603H1 contained a cDNA fragment of about 2.0 kb as inserted thereinand pKM603L1 a cDNA fragment of about 0.9 kb as inserted therein.

6. Base Sequences of the Variable Regions in the H Chain cDNA and LChain cDNA

The base sequences of the variable regions in the H chain cDNA and Lchain cDNA obtained as mentioned above in Paragraphs 4 and 5 weredetermined by the dideoxy method [Maniatis et al. (ed.): MolecularCloning, 13.42, Cold Spring Harbor Laboratory, 1989] using SequenaseVersion 2.0 DNA Sequencing Kit manufactured by United States BiochemicalCorporation. All the cDNA had a methionine codon, presumably theinitiation codon ATG, at the 5' terminus and were leadersequence-containing full-length cDNAs. Based on the base sequences ofthe respective cDNAs, the amino acid sequences of the H chain and Lchain of KM-796, KM-750 and KM-603 were deduced. The amino acid sequenceof the KM-796 H chain is shown in SEQ ID NO:1, that of the L chain ofKM-796 and KM-750 in SEQ ID NO:2, that of the KM-750 H chain in SEQ IDNO:3, that of the KM-603 H chain in SEQ ID NO:4 and that of the KM-603 Lchain in SEQ ID NO:5.

7. Construction of KM-796- and KM-750-derived Chimeric Human Antibody HChain and L Chain Expression Vvectors

(1) Construction of a Vector, pAGE147, Carrying the Moloney MouseLeukemia Virus Terminal Repeat Promoter/Enhancer

The plasmid pPMOL1 (2 μg), described in JP-A-1-63394, was dissolved in30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mMmagnesium chloride and 6 mM 2-mercaptoethanol, 20 units of SmaI wasadded, and digestion was carried out at 30° C. for 3 hours. Then, sodiumchloride was added to a concentration of 50 mM, 20 units of ClaI wasadded, and digestion was conducted at 37° C. for 2 hours. The reactionmixture was subjected to agarose gel electrophoresis, and a DNA fragment(about 0.6 kb) containing the Moloney mouse leukemia virus terminalrepeat promoter/enhancer was recovered.

Then, the following two synthetic DNAs were synthesized using anautomatic DNA synthesizer (model 380A manufactured by Applied BiosystemsCo., Ltd.). ##STR1##

The thus-obtained synthetic DNAs (25 picomoles each) were dissolved in10 μl of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mMmagnesium chloride, 5 mM DTT (dithiothreitol), 0.1 mM EDTA and 0.5 mMadenosine triphosphate (hereinafter, "ATP"), 5 units of T4 DNA kinasewas added, and 5'-phosphorylation was carried out at 37° C. for 30minutes.

The plasmid pPMOL1-derived ClaI-SmaI fragment (0.6 kb, 0.05 μg) and two5' -phosphorylated synthetic DNAs (1 picomole each), obtained asdescribed above, and a HindIII linker (5'-pCAAGCTTG-3'; Takara Shuzo) (1picomole) were dissolved in 30 μl of 66 mM Tris-hydrochloride buffer (pH7.5) containing 6.6 mM magnesium chloride, 10 mM DTT and 0.1 mM ATP, 200units of T4 DNA ligase (Takara Shuzo; hereinafter the same shall apply)were added, and ligation was carried out at 12° C. for 16 hours. Theresultant DNA fragment was recovered by ethanol precipitation anddissolved in 20 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride, 100 mM sodium chloride and 6 mM2-mercaptoethanol, 10 units of HindIII and 10 units of XhoI were added,and digestion was carried out at 37° C. for 2 hours. The reaction wasterminated by phenol-chloroform extraction, and the DNA fragment wasrecovered by ethanol precipitation.

Separately, 1 μg of the plasmid pAGE107 [Cytotechnology, 3, 133 (1990)]was dissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride, 100 mM sodium chloride and 6 mM2-mercaptoethanol, 10 units of HindIII and 10 units of XhoI were added,and digestion was carried out at 37° C. for 2 hours. The reactionmixture was subjected to agarose gel electrophoresis, and a DNA fragment(about 6.0 kb) containing the G418 resistance gene and ampicillin(hereinafter, "Ap") resistance gene was recovered.

The plasmid pAGE107-derived HindIII-XhoI fragment (6.0 kb, 0.3 μg) andplasmid pPMOL1-derived HindIII-XhoI fragment (0.63 kb, 0.01 μg) obtainedas mentioned above were dissolved in 20 μl of 66 mM Tris-hydrochloridebuffer (pH 7.5) containing 6.6 mM magnesium chloride, 10 mM DTT and 0.1mM ATP, 200 units of T4 DNA ligase were added, and ligation was carriedout at 12° C. for 16 hours. The thus-obtained recombinant plasmid DNAwas used to transform Escherichia coli HB101, and the plasmid pAGE147shown in FIG. 4 was obtained.

(2) Construction of a Vector, pAGE148, Carrying the β-globin 3' SplicingSignal (SP)

For introducing the β-globin 3' splicing signal into the chimeric humanantibody expression vector at a site downstream from the antibodyconstant region gene, a vector (pAGE148), was constructed as follows,which contained the β-globin 3' splicing signal and the same genes asthose in the chimeric human antibody expression vector (except for thehuman antibody constant region gene).

Two μg of pSE1UK1SEd1-3, described in JP-A-2-257851, were added to 30 μlof 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT. After addition of 10 unitsof HindIII, digestion was carried out at 37° C. for 4 hours. Thereaction mixture was subjected to phenol-chloroform extraction and thento ethanol precipitation. The precipitate was dissolved in 20 μl of DNApolymerase I buffer, 5 units of Escheichia coli-derived DNA polymerase IKlenow fragment were added, and the 5' cohesive ends produced by HindIIIdigestion were rendered blunt by incubation at 22° C. for 30 minutes.The reaction mixture was subjected to phenol-chloroform extraction andthen to ethanol precipitation, 30 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT and 10 unitsof KpnI were added, and digestion was effected at 37° C. for 4 hours.The reaction mixture was subjected to phenol-chloroform extraction andthen to ethanol precipitation, 30 μl of 50 mM Tris-hydrochloride buffer(pH 7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and1 mM DTT and 10 units of XhoI were added, and digestion was carried outat 37° C. for 4 hours. The reaction mixture was fractionated by agarosegel electrophoresis and two DNA fragments, about 6.67 kb and about 1.98kb in size, were recovered (about 0.2 μg each).

Then, 2 μg of pAGE147 obtained in (1) was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of KpnI was added, and digestion was effected at37° C. for 4 hours. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, 30 μl of50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT and 10 units of XhoI wereadded, and digestion was carried out at 37° C. for 4 hours. The reactionmixture was fractionated by agarose gel electrophoresis and about 0.2 μgof a DNA fragment of about 0.66 kb was recovered.

Then, 0.1 μg of the XhoI-HindIII fragment (about 6.67 kb) ofpSE1UK1SEd1-3, as obtained above, 0.1 μg of the KpnI-HindIII fragment(about 1.98 kb), obtained above, and 0.1 μg of the XhoI-KpnI fragment(about 0.66 kb) of pAGE147, as obtained above, were dissolved in a totalof 20 μl of T4 ligase buffer. Three hundred fifty units of T4 ligasewere added to the solution, and ligation was carried out at 4° C. for 24hours. The thus-obtained recombinant plasmid DNA was used to transformEscherichia coli HB101, and the plasmid pAGE148 shown in FIG. 5 wasobtained.

(3) Construction of KM-796- and KM-750-Derived Chimeric Human Antibody HChain Expression Vectors

First, the cDNA coding for the antibody variable region in the plasmidpKM796H1 or pKM750H1 was excised by cleavage at the 5' -terminal EcoRIsite and the MaeIII site near the 3' end of said cDNA and joined,together with a synthetic DNA having the base sequence shown in SEQ IDNO:12, to the chimeric human antibody H chain expression vectorpChi641HAM1, as follows (FIG. 6).

Three μg of pKM796H1 or pKM750H1, obtained in Paragraph 4, were added to30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride, 100 mM sodium chloride and 1 mM DTT. Further, 10units of EcoRI and 10 units of MaeIII were added, and digestion waseffected at 37° C. for 4 hours. The reaction mixture was fractionated byagarose gel electrophoresis and about 0.3 μg of a DNA fragment of about0.43 kb was recovered. Then, 3 μg of pChi641HAM1, obtained in ReferenceExample 2, was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of EcoRIand 10 units of ApaI were also added, and digestion was carried out at37° C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.0 μg of a DNA fragment of about 9.0 kb wasrecovered. Then, 0.1 μg of the EcoRI-MaeIII fragment (about 0.43 kb) ofpKM796H1 or pKM750H1, as obtained above, 0.1 μg of the EcoRI-ApaIfragment (about 9.0 kb) of pChi641HAM1, as obtained above, and 0.3 μg ofa synthetic DNA having the base sequence shown in SEQ ID NO:12 weredissolved in a total of 20 μl of T4 ligase buffer, 350 units of T4ligase was further added to the solution, and ligation was carried outat 4° C. for 24 hours. The thus-obtained recombinant plasmid DNA wasused to transform Escherichia coli HB101. In this way, the plasmidspChi796HM1 and pChi750HM1, shown in FIG. 6, were obtained.

Then, the β-globin 3' splicing signal was introduced into the plasmidspChi796HM1 and pChi750HM1 by the method described below to constructKM796- and KM-750-derived chimeric human antibody H chain expressionvectors (FIG. 7).

Three μg of pChi796HM1 or pChi750HM1 were added to 30 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 0.01% bovine serum albumin(hereinafter, "BSA"). Ten units of XhoI and 10 units of KpnI were alsoadded, and digestion was carried out at 37° C. for 4 hours. The reactionmixture was fractionated by agarose gel electrophoresis and about 0.3 μgof a DNA fragment of about 3.4 kb was recovered. Then, 3 μg of pAGE148obtained in (2) was added to 30 μl of 33 mM Tris-acetate buffer (pH 7.9)containing 10 mM magnesium acetate, 66 mM potassium acetate, 0.5 mM DTTand 0.01% BSA; 10 units of XhoI and 10 units of KpnI were further added,and digestion was effected at 37° C. for 4 hours. The reaction mixturewas fractionated by agarose gel electrophoresis and about 0.3 μg of aDNA fragment of about 8.7 kb was recovered. Then, 0.1 μg of theXhoI-KpnI fragment of pChi796HM1 or pKM750HM1 and 0.1 μg of theXhoI-KpnI fragment of pAGE148 were dissolved in a total of 20 μl of T4ligase buffer, 350 units of T4 ligase was further added to the solution,and ligation was carried out at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101.The plasmids pChi796HMS1 and pChi750HMS1 shown in FIG. 7 were thusobtained.

(4) Construction of KM-796- and KM-750-Derived Chimeric Human Antibody LChain Expression Vectors

First, the cDNA coding for the antibody variable region in the plasmidpKM796L1 or pKM750L1 was excised by cleavage at the 5' -terminal EcoRIsite and the Af1III site near the 3' end of said cDNA and joined,together with a synthetic DNA having the base sequence shown in SEQ IDNO:13, to the chimeric human antibody L chain expression vectorpChiIgLA1, as follows (FIG. 8).

Three μg of pKM796L1 or pKM750L1 were added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Further, 10 units of EcoRI and 10units of Af1III were added, and digestion was effected at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 0.39 kb wasrecovered. Then, 3 μg of pChiIgLA1 obtained in Reference Example 1 wasadded to 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units ofEcoRI and 10 units of EcoRV were further added, and digestion wascarried out at 37° C. for 4 hours. The reaction mixture was fractionatedby agarose gel electrophoresis and about 1 μg of a DNA fragment of about8.6 kb was recovered.

Then, 0.1 μg of the EcoRI-Af1III fragment of pKM796L1 or pKM750L1, asobtained above, 0.1 μg of the EcoRI-EcoRV fragment of pChiIgLA1, asobtained above, and 0.3 μg of a synthetic DNA, having the base sequenceshown in SEQ ID NO:13, were dissolved in a total of 20 μl of T4 ligasebuffer; 350 units of T4 ligase was further added to the solution, andligation was carried out at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101. Inthis way, the plasmids pChi796LI1 and pChi750LI1 shown in FIG. 8 wereobtained.

Then, the Moloney mouse leukemia virus terminal repeat promoter/enhancerwas introduced into the plasmids pChi796LI1 and pChi750LI1 in thefollowing manner (FIG. 9).

Three μg of pChi796LI1 and pChi750LI1 were added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Further, 10 units of EcoRI and 10units of XhoI were added, and digestion was effected at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 8.2 kb wasrecovered Then, 3 μg of the chimric human antibody H chain expressionvector pChi641HAM1 obtained in Reference Example 2 was added to 30 μl of50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10units of XhoI were further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 0.6 kb wasrecovered.

Then, 0.1 μg of the EcoRI-XhoI fragment of pChi796LI1 or pKM750LI1 asobtained above and 0.1 μg of the EcoRI-XhoI fragment of pChi641HAM1 asobtained above were dissolved in a total of 20 μl of T4 ligase buffer,350 units of T4 ligase was further added to the solution, and ligationwas carried out at 4° C. for 24 hours. The thus-obtained recombinantplasmid DNA was used to transform Escherichia coli HB101. In this way,the plasmids pChi796LM1 and pChi750LM1 shown in FIG. 9 were obtained.

Then, the β-globin 3' splicing signal was introduced into the plasmidspChi796LM1 and pChi750LM1 in the manner mentioned below to constructKM-796- and KM-750-derived chimeric human antibody L chain expressionvectors (FIG. 10).

Three μg of pChi796LM1 or pChi750LM1 were added to 30 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 0.01% BSA. Further, 10 units of XhoIand 10 units of KpnI were added, and digestion was carried out at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 2.0 kb wasrecovered. Then, 3 μg of pAGE148 obtained in (2) was added to 30 μl of33 mM Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate,66 mM potassium acetate, 0.5 mM DTT and 0.01% BSA; 10 units of XhoI and10 units of KpnI were further added, and digestion was carried out at37° C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 8.7 kb wasrecovered. Then 0.1 μg of the XhoI-KpnI fragment of pChi796LM1 orpKM750LM1 as obtained above and 0.1 μg of the XhoI-KonI fragment ofpAGE148 were dissolved in a total of 20 μl of T4 ligase buffer, 350units of T4 ligase was further added, and ligation was carried out at 4°C. for 24 hours. The thus-obtained recombinant plasmid DNA was used totransform Escherichia coli HB101. In this way, the plasmids pChi796LMS1and pChi750LMS1 shown in FIG. 10 were obtained.

8. Construction of KM-796- and KM-750-Derived Chimeric Human Antibody HChain and L Chain Tandem Expression Vectors

Tandem expression vectors containing the chimeric human antibody Hchain-encoding cDNA and L chain-encoding cDNA on one and the same vectorwere constructed (FIG. 11 and FIG. 12).

Three μg of pChi796HMS1 or pChi750HMS1, obtained in Paragraph 7, wereadded to 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT. Further, 10units of MluI and 10 units of SaIl were added, and digestion was carriedout at 37° C. for 4 hours. The reaction mixture was fractionated byagarose gel electrophoresis. In each case, about 0.3 μg of a DNAfragment of about 5.9 kb was recovered. Then, 2 μg of pAGE107 describedin EP-A-0 405 285 was dissolved in 30 μl of 10 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride, 50 mM sodiumchloride and 1 mM DTT; 10 units of MluI and 10 units of SalI werefurther added, and digestion was carried out at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.2 μg of a DNA fragment of about 3.55 kb was recovered. Then, 0.1μg of the MluI-SalI fragment of pChi796HMS1 or pChi750HMS1 and 0.1 μg ofthe MluI-SalI fragment of pAGE107 were dissolved in a total of 20 μl ofT4 ligase buffer, 350 units of T4 ligase was added, and ligation wascarried out at 4° C. for 24 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 to give the plasmidpChi796H107 or pChi750H107 shown in FIG. 11.

Then, 3 μg of pChi796H107 or pChi750H107 was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,50 mM sodium chloride and 1 mM DTT, 10 units of ClaI was further added,and digestion was carried out at 37° C. for 4 hours. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation. The precipitate was dissolved in 20 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the mixture was incubated at 22° C. for30 minutes for rendering the cohesive ends formed upon ClaI digestionblunt-ended. The reaction mixture was further subjected tophenol-chloroform extraction and then to ethanol precipitation. To theprecipitate were added 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT,and 10 units of MluI. Digestion was carried out at 37° C. for 4 hoursand the reaction mixture was fractionated by agarose gelelectrophoresis. In each case, about 0.3 μg of a DNA fragment of about7.5 kb was recovered. Then, 3 μg of pChi796LMS1 or pChi750LMS1 was addedto 30 μl of 20 mM Tris-hydrochloride buffer (pH 8.5) containing 10 mMmagnesium chloride, 100 mM potassium chloride and 1 mM DTT, 10 units ofXhoI was further added, and digestion was carried out at 37° C. for 4hours. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation. The precipitate wasdissolved in 20 μl of DNA polymerase I buffer, 5 units of Escherichiacoli-derived DNA polymerase I Klenow fragment was added, and the mixturewas incubated at 22° C. for 30 minutes for rendering the cohesive endsformed upon XhoI digestion blunt-ended. The reaction mixture was furthersubjected to phenol-chloroform extraction and then to ethanolprecipitation. To the precipitate was added 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,50 mM sodium chloride and 1 mM DTT as well as 10 units of MluI.Digestion was carried out at 37° C. for 4 hours and the reaction mixturewas fractionated by agarose gel electrophoresis. In each case, about 0.3μg of a DNA fragment of about 9.3 kb was recovered. Then, 0.1 μg of theMluI-ClaI fragment of pChi796H107 or pChi750H107, as obtained above, and0.1 μg of the MluI-XhoI fragment of pChi796LMS1 or pChi750LMS1, asobtained above, were dissolved in a total of 20 μl of T4 ligase buffer;350 units of T4 ligase was further added, and ligation was carried outat 4° C. for 24 hours. The thus-obtained recombinant plasmid DNA wasused to transform Escherichia coli HB101, and the plasmid pChi796HL1 orpChi750HL1 shown in FIG. 12 was obtained.

9. Construction of a KM-603-Derived Chimeric Human Antibody H ChainExpression Vector

First, the antibody variable region-encoding cDNA of the plasmidpKM603H1 was excised by cleavage at the 5'-terminal EcoRI site and theStyI site near the 3' end of said cDNA and joined to the chimeric humanantibody H chain expression vector pChi641HAM1 together with a syntheticDNA having the base sequence shown in SEQ ID NO:14 in the followingmanner (FIG. 13).

Three μg of pKM603H1 obtained in Paragraph 5 were added to 30 μl of 50mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT, followed by furtheraddition of 10 units of EcoRI and 10 units of StyI. Digestion wascarried out at 37° C. for 4 hours. The reaction mixture was fractionatedby agarose gel electrophoresis and about 0.3 μg of a 0.4-kb DNA fragmentwas recovered. Then, 3 μg of pChi641HAM1, obtained in Reference Example2, was added to 30 μl of 10 mM Tris-hydrochloride (pH 7.5) containing 10mM magnesium chloride and 1 mM DTT, 10 units of EcoRI and 10 units ofApaI were further added, and digestion was effected at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.0 μg of a DNA fragment of about 9.0 kb wasrecovered. Then, 0.1 μg of the EcoRI-StyI fragment (about 0.4 kb) ofpKM603H1, as obtained above, and 0.1 μg of the EcoRI-ApaI fragment(about 9.0 kb) of pChi641HAM1, as obtained above, were dissolved,together with 0.3 μg of a synthetic DNA having the base sequence shownin SEQ ID NO:14, in a total of 20 μl of T4 ligase buffer; 350 units ofT4 ligase was added to the solution, and ligation was effected at 4° C.for 24 hours. The thus-obtained recombinant plasmid DNA was used totransform Escherichia coli HB101 and the plasmid pChi603HM1 shown inFIG. 13 was obtained.

Then, a KM-603-derived chimeric human antibody H chain expression vectorwas constructed by introducing the β-globin 3' splicing signal into theplasmid pChi603HM1 in the following manner (FIG. 14).

Three μg of pChi603HM1 obtained above were added to 30 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 0.01% BSA. Further, 10 units of XhoIand 10 units of KpnI were added, and digestion was carried out at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 3.3 kb wasrecovered.

Then, 3 μg of pAGE148 obtained in Paragraph 7 (2) was added to 30 μl of33 mM Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate,66 mM sodium acetate, 0.5 mM DTT and 0.01% BSA; 10 units of XhoI and 10units of KvnI were further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 8.7 kb wasrecovered. Then, 0.1 μg of the XhoI-KpnI fragment of pChi603HM1, asobtained above, and 0.1 μg of the XhoI-KpnI fragment of pAGE148, asobtained above, were dissolved in a total of 20 H1 of T4 ligase buffer;350 units of T4 ligase was added to the solution, and ligation wascarried out at 4° C. for 24 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 and the plasmidpChi603HMS1 shown in FIG. 14 was obtained.

10. Construction of a KM-603-Derived Chimeric Human Antibody L ChainExpression Vector

First, the antibody variable region cDNA in the plasmid pKM603L1 wasexcised by cleavage at the 5' terminal EcoRI site and the AflIII sitenear the 3' end and joined to the chimeric human antibody L chainexpression vector pChiIgLA1 together with a synthetic DNA having thebase sequence defined by SEQ ID NO:15 (FIG. 15).

Thus, 3 μg of pKM603L1 obtained in Paragraph 5 was added to 30 μl of 50mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10units of AflIII were further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 0.4 kb wasrecovered. Then, 3 fg of pChiIgLA1 obtained in Reference Example 1 wasadded to 30 4l of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units ofEcoRI and 10 units of EcoRV were further added, and digestion wascarried out at 37° C. for 4 hours. The reaction mixture was fractionatedby agarose gel electrophoresis and about 1 μg of a DNA fragment of about8.6 kb was recovered. Then, 0.1 μg of the EcoRI-Af1III fragment ofpKM603L1, as obtained above, 0.1 μg of the EcoRI-EcoRV fragment ofpChiIgLA1, as obtained above, and 0.3 μg of a synthetic DNA, having thebase sequence defined by SEQ ID NO:15, were dissolved in a total of 20μl of T4 ligase buffer; 350 units of T4 ligase was added to thesolution, and ligation was carried out at 4° C. for 24 hours. Thethus-obtained recombinant plasmid DNA was used to transform Escherichiacoli HB101 and the plasmid pChi603LI1 shown in FIG. 15 was obtained.

Then, the Moloney mouse leukemia virus terminal repeat promoter/enhancerwas introduced into the plasmid pChi603LI1 in the following manner (FIG.16).

Thus, 3 μg of pChi603LI1 obtained above was added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10 units ofXhoI were further added, and digestion was carried out at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 8.3 kb wasrecovered. Then, 3 μg of pChi641HAM1 obtained in Reference Example 2 wasadded to 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units ofEcoRI and 10 units of XhoI were further added, and digestion waseffected at 37° C. for 4 hours. The reaction mixture was fractionated byagarose gel electrophoresis and about 0.3 μg of a DNA fragment of about0.6 kb was recovered. Then, 0.1 μg of the EcoRI-XhoI fragment ofpChi603LI1, as obtained above, and 0.1 μg of the EcoRI-XhoI fragment ofpChi641HAM1, as obtained above, were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 ligase was added to the solution, andligation was effected at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Eschrichia coli HB101 togive the plasmid pChi603LM1 shown in FIG. 16.

A KM-603-derived chimeric human antibody L chain expression vector wasthen constructed by introducing the β-globin 3' splicing signal into theplasmid pChi603LM1, as follows (FIG. 17).

Thus, 3 μg of pChi603LM1 obtained above was added to 30 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 0.01% BSA, 10 units of XhoI and 10units of KpnI were further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 2.0 kb wasrecovered. Then, 3 μg of pAGE148 obtained in Paragraph 7 (2) was addedto 30 μl of 33 mM Tris-acetate buffer (pH 7.9) containing 10 mMmagnesium acetate, 66 mM potassium acetate, 0.5 mM DTT and 0.01% BSA, 10units of XhoI and 10 units of KpnI were further added, and digestion waseffected at 37° C. for 4 hours. The reaction mixture was fractionated byagarose gel electrophoresis and about 0.3 μg of a DNA fragment of about8.7 kb was recovered. Then, 0.1 μg of the XhoI-KpnI fragment ofpChi603LM1, as obtained above, and 0.1 μg of the XhoI-KpnI fragment ofpAGE148, as obtained above, were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 ligase was added to the solution, andligation was carried out at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 togive the plasmid pChi603LMS1 shown in FIG. 17.

11. Expression of the KM-796- and KM-750-Derived Chimeric Human Anti-GM₂Antibody in YB2/0 Cells

The plasmids were introduced into YB2/0 cells by the electroporationmethod of Miyaji et al. [Cytotechnbology, 3, 133-140 (1990)].

After introduction of 4 μg of pChi750HL1 or pChi796HL1 obtained inParagraph 8 into 4×10⁶ YB2/0 (ATCC CRL1581) cells, the cells weresuspended in 40 ml of RPMI-1640-FCS(10) [RPMI1640 medium (NissuiPharmaceutical) containing 10% of FCS, 1/40 volume of 7.5% NaHCO₃, 3% of200 mM L-glutamine solution (Gibco) and 0.5% of penicillin-streptomycinsolution (Gibco; containing 5,000 units/ml penicillin and 5,000 μg/mlstreptomycin)], and the suspension was distributed in 200-μl portionsinto wells of 96-well microtiter plates. After 24 hours of incubation at37° C. in a CO₂ incubator, G418 (Gibco) was added to a concentration of0.5 mg/ml and then incubation was continued for 1 to 2 weeks.Transformant colonies appeared, the culture fluid was recovered fromeach well in which the cells had grown to confluence and anenzyme-linked immunosorbent assay (ELISA) was conducted for anti-GM₂chimeric human antibody activity measurement.

Enzyme-Linked Immunosorbent Assay (ELISA)

In a solution of 5 ng of phosphatidylcholine (Sigma) and 2.5 ng ofcholesterol (Sigma) in 2 ml of ethanol was dissolved 2 ng of GM₂(N-acetyl-GM₂ ; Boehringer Mannheim) or some other ganglioside. Thesolution or dilutions thereof were respectively distributed in 20-μlportions into wells of 96-well microtiter plates (Greiner) and, afterair drying, blocking was effected with PBS containing 1% BSA. Eachculture supernatant for each transformant, each purified mousemonoclonal antibody solution and each purified chimeric human antibodysolution were distributed in 50- to 100-μl portions into the wells andthe reaction was allowed to proceed at room temperature for 1 to 2hours. The wells were then washed with PBS, and 50 to 100 μl ofperoxidase-labeled antibody were added thereto followed by reaction atroom temperature for 1 to 2 hours. The wells were washed with PBS and anABTS substrate solution [prepared by dissolving 550 mg of2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in0.1M citrate buffer (pH 4.2) and adding, just prior to use, hydrogenperoxide to a concentration of 1 μl/ml] was added in 50- to 100-μlportions to each well for color development, followed by OD₄₁₅measurement.

The clone showing the highest activity in ELISA among the clonesobtained gave a chimeric human anti-GM₂ antibody content of about 1.0μg/ml of culture fluid.

Cells of the clone showing the above-mentioned chimeric human anti-GM₂antibody activity were suspended in RPMI1640-FCS(10) medium containing0.5 mg/ml G418 and 50 nM methotrexate (hereinafter, "MTX") to aconcentration of 1 to 2×10⁵ cells/ml, and the suspension was distributedin 2-ml portions into wells of 24-well plates. Incubation was performedat 37° C. in a CO₂ incubator for 1 to 2 weeks to induce 50 nMMTX-resistant clones. At the time of confluence, the chimeric humananti-GM₂ antibody activity in each culture fluid was determined byELISA. The 50 nM MTX-resistant clone showing the highest activity amongthe clones obtained showed a chimeric human anti-GM₂ antibody content ofabout 5.0 μg/ml.

Cells of the above 50 nM MTX-resistant clone were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml, and the suspension was distributedin 2-ml portions into wells of 24-well plates. Incubation was carriedout at 37° C. in a CO₂ incubator for 1 to 2 weeks to induce 200 nMMTX-resistant clones. At the time of confluence, each culture fluid wasassayed for chimeric human anti-GM₂ antibody activity by ELISA. The 200nM MTX-resistant clone showing the highest activity among the clonesobtained had a chimeric human anti-GM₂ antibody content of about 10μg/ml. The 200 nM MTX-resistant clones obtained from pChi750HL1 andpChi796HL1 were named transformants "KM966"(KM-796-derived chimerichuman antibody KM966-producing strain) and "KM967" (KM-750-derivedchimeric human antibody KM967-producing strain), respectively.

The following SDS-polyacrylamide gel electrophoresis (SDS-PAGE)confirmed that the above transformants KM966 and KM967 express therespective chimeric human anti-GM₂ antibodies.

The transformants KM966 and KM967 were each suspended in GIT medium(Nippon Pharmaceutical) containing 0.5 mg/ml G418 and 200 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml. Each suspension was distributed in100-ml portions into 175 cm² flasks (Greiner). Cultivation was performedat 37° C. in a CO₂ incubator for 5 to 7 days. At the time of confluence,the culture fluid was recovered. Treatment of about 1 liter of theculture fluid with Affi-Gel Protein A MAPS-II kit (Bio-Rad) gave about 5mg of a purified chimeric human anti-GM₂ antibody for each transformant.About 2 μg of the purified chimeric human anti-GM₂ antibody KM966 orKM967 was electrophoresed by the conventional method [Laemmli: Nature,227, 680 (1970)] for molecular weight checking. The results are shown inFIG. 18. As shown in FIG. 18, both KM966 and KM967 gave an antibody Hchain molecular weight of about 50 kilodaltons and an antibody L chainmolecular weight of about 25 kilodaltons under reducing conditions,indicating the correctness in molecular weight of the H chain and Lchain expressed. For each of KM966 and KM967, the molecular weight ofthe chimeric human antibody under nonreducing conditions was about 150kilodaltons, indicating that the antibody expressed was composed of twoH chains and two L chains and was correct in size.

12. Expression of KM-603-Derived Chimeric Human Anti-GM₂ Antibodies inSP2/0 Cells

A 2-μg portion of the plasmid pChi603HMS1 or pChi603LMS1 obtained inParagraph 9 was introduced into 4×10⁶ cells of YB2/0 (ATCC CRL1581) byelectroporation in the same manner in Paragraph 11. The cells weresuspended in 40 ml of RPMI1640-FCS(10) and the suspension wasdistributed in 200-μl portions into wells of 96-well microtiter plates.After 24 hours of incubation in a CO₂ incubator at 37° C., G418 (Gibco)was added to a concentration of 0.5 mg/ml and incubation was continuedfor 1 to 2 weeks. Transformant colonies appeared. The culture fluid wasrecovered from confluent wells and the chimeric human anti-GM₂ antibodyactivity was measured by ELISA as described above. The clone showing thehighest chimeric human anti-GM₂ antibody activity among the clonesobtained gave a chimeric human anti-GM₂ antibody content of about 0.1μg/ml of culture fluid.

Cells of the clone showing the above-mentioned chimeric human anti-GM₂antibody activity were suspended in RPMI1640-FCS(10) medium containing0.5 mg/ml G418 and 50 nM MTX to a concentration of 1 to 2×10⁵ cells/mland the suspension was distributed in 2-ml portions into wells of24-well plates. Clones resistant to 50 nM MTX were induced by incubatingin a CO₂ incubator at 37° C. for 2 to 3 weeks. When confluence wasattained, the culture fluids were subjected to ELISA for chimeric humananti-GM₂ antibody activity measurement. Among the 50 nM MTX-resistantclones obtained, the clone showing the highest activity gave a chimerichuman anti-GM₂ antibody content of about 0.3 μg/ml of culture fluid.

Cells of the above 50 nM MTX-resistant clone were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml and the suspensions as distributedin 2-ml portions into well of 24-well plates. Clones resistant to 200 nMMTX were induced by following incubation in a CO₂ incubator at 37° C.for 2 to 3 weeks. When confluence was attained, the chimeric humananti-GM₂ antibody activity in the culture fluid was measured by ELISA.Among the 200 nM MTX-resistant clones obtained, the clone showing thehighest activity gave a chimeric human anti-GM₂ antibody content ofabout 0.5 μg/ml of culture fluid.

Cells of the above 200 nM MTX-resistant clone were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 500 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml and the suspension was distributedin 2-ml portions into well of 24-well plates. Clones resistant to 500 nMMTX were induced following incubation in a CO₂ incubator at 37° C. for 1to 2 weeks. When confluence was attained, the chimeric human anti-GM₂antibody activity in the culture fluid was determined by ELISA. Amongthe 500 nM MTX-resistant clones obtained, the one showing the highestactivity gave a chimeric human anti-GM₂ antibody content of about 1.0μg/ml of culture fluid. This 500 nM MTX-resistant clone was named"transformant KM968".

The following SDS-PAGE confirmed the expression of a chimeric humananti-GM₂ antibody in the above transformant KM968.

Cells of the transformant KM968 were suspended in GIT medium (NipponPharmaceutical) containing 0.5 mg/ml G418 and 500 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml and the suspension was distributedin 100-ml portions into 175 cm² flasks (Greiner). Cultivation wasconducted in a CO₂ incubator at 37° C. for 5 to 7 days and, whenconfluence was attained, the culture fluid was recovered. Treatment ofabout 3.0 liters of the culture fluid with Affi-Gel Protein A MAPS-IIkit (Bio-Rad) gave about 1 mg of a purified chimeric human anti-GM₂antibody. About 2 μg of this purified chimeric human anti-GM₂ antibodyKM968 was electrophoresed by the conventional method [Laemmli: Nature,227, 680 (1970)] for molecular weight checking. The results are shown inFIG. 19. Under reducing conditions, the molecular weight of the antibodyH chain was about 50 kilodaltons and the molecular weight of theantibody L chain was about 25 kilodaltons, thus confirming theexpression of the H chain and L chain having the correct molecularweight. Under nonreducing conditions, the molecular weight of thechimeric human antibody was about 150 kilodaltons, confirming that theantibody expressed was composed of two H chains and two L chains and wascorrect in size.

13. Reaction Specificity of the Chimeric Human Anti-GM₂ Antibodies

The reactivities of the chimeric anti-GM₂ antibodies with gangliosideGM₁, N-acetyl-GM₂ (Boehringer Mannheim), N-glycolyl-GM₂, N-acetyl-GM₃,N-glycolyl-GM₃, GD_(1a), GD_(1b) (Iatron), GD₂, GD₃ (Iatron) and GQ_(1b)(Iatron) were examined by the technique of ELISA. The results are shownbelow in Table 1. GM₁ and GD_(1a) were purified from the bovine brain,N-glycolyl-GM₂ and N-glycolyl-GM₃ from the mouse liver, N-acetyl-GM₃from canine erythrocytes, and GD₂ from the cultured cell line IMR32(ATCC CCL127), by a known method [J. Biol. Chem., 263, 10915 (1988)].

As shown in Table 1, it was confirmed that the chimeric human anti-GM₂antibodies KM966 and KM967 specifically react with GM₂. The reactivityof KM966 was greater than that of KM967, however. On the contrary, KM968(KM-603-derived chimeric human antibody) did not show any reactivity forGM₂.

                  TABLE 1                                                         ______________________________________                                                    Binding activity of antibody (OD.sub.415)                         Ganglioside   KM966 (5 μg/ml)                                                                        KM967 (5 μg/ml)                                  ______________________________________                                        GM.sub.1      0.105       0.000                                               N-Acetyl-GM.sub.2                                                                           0.870       0.423                                               N-Glycolyl-GM.sub.2                                                                         0.774       0.065                                               N-Acetyl-GM.sub.3                                                                           0.002       0.000                                               N-Glycolyl-GM.sub.3                                                                         0.122       0.001                                               GD.sub.1a     0.004       0.000                                               GD.sub.1b     0.002       0.000                                               GD.sub.2      0.095       0.001                                               GD.sub.3      0.004       0.000                                               GQ.sub.1b     0.005       0.000                                               ______________________________________                                    

14. Reactivities of the Chimeric Human Anti-GM₂ Antibodies KM966 andKM967 with Cancer Cells (Fluorescent Antibody Technique)

Suspended in PBS were 1×10⁶ cells of cultured human lung small cellcarcinoma cell line QC90 [Cancer Res., 49, 2683 (1989)], NCI-H69 (ATCCHTB119), NCI-H128 (ATCC HTB120), SBC-1 (JCRB 0816), SBC-2 (JCRB 0817),SBC-3 (JCRB 0818), SBC-5 (JCRB 0819), RERF-LC-MA (JCRB 0812), Lu-134-A-H(JCRB 0235), Lu-139 (RCB 469), Lu-130 (RCB 465), Lu-135 (RCB 468),Lu-134-B (RCB 467), Lu-140 (RCB 470), PC-6 [Naito et al.: Gann to KagakuRyoho (Cancer and Chemotherapy), 5 (suppl.), 89 (1978)], cultured humanlung squamous carcinoma cell line PC-1 [Naito et al.: Gann to KagakuRyoho, 5 (suppl.), 89 (1978)], PC-10 [Naito et al.: Gann to KagakuRyoho, 5 (suppl.), 89 (1978)], Colo16 [Moor et al.: Cancer Res., 35,2684 (1975)], Calu-1 (ATCC HTB54), SK-LC-4 [Proc. Natl. Acad. Sci.U.S.A., 85, 4441 (1988)], cultured human lung adenocarcinoma cell linePC-7 [Hayata et al.: Hito Gansaibo no Baiyo (Human Cancer Cell Culture),131 (1975)], PC-9 [Kinjo et al.: Brit. J. Cancer, 39, 15 (1979)], PC-12(ATCC CRL1721), RERF-LC-MS (JCRB 0081), HLC-1 (RCB 083), cultured humanlung large cell carcinoma cell line PC-13 [Oboshi et al.: Tanpakushitsu,Kakusan, Koso (Protein, Nucleic Acid, Enzyme), 23, 697 (1978)], Lu65(JCRB 0079), CALU-6 (ATCC HTB56), SK-LC-6 [Proc. Natl. Acad. Sci.U.S.A., 85, 4441 (1988)], cultured human neuroblastoma cell line YT-nu[Ishikawa et al.: Acta Path. Jap., 27, 697 (1977)], NAGAI [Ishikawa etal.: Acta Path. Jap., 29, 289 (1979)], NB-1 [Ishikawa et al.: Acta Path.Jap., 27, 697 (1977)], IMR32 (ATCC CCL127), GOTO (JCRB 0612), NB-9 (RCB477), SK-N-MC (ATCC HTB10), cultured human brain tumor (glioma) cellline SK-MG-4 [EMBO J., 6, 2939 (1987)], A172 (ATCC CRL1620), T98G (ATCCCRL1690), U-118MG (ATCC HTB15), cultured human leukemia cell line HSB-2(ATCC CCL120.1), ATN-1, U-937 (ATCC CRL1593), HPB-ALL [Oboshi et al.:Tanpakushitsu, Kakusan, Koso, 23, 697 (1978)], CCRF-SB (ATCC CCL120),KOPN-K [Hanei et al.: Haigan (Lung Cancer), 25, 524 (1985)], TYH[Haranaka et al.: Int. J. Cancer, 36, 313 (1985)], MOLT-3 (ATCCCRL1552), CCRF-CEM (ATCC CCL119), TALL-1 (JCRB 0086), NALL-1 [Oboshi etal.: Tanpakushitsu, Kakusan, Koso, 23, 697 (1978), CCRF-SB (JCRB 0032),THP-1 (ATCC TIB202), HEL92-1-7 (ATCC TIB180), cultured human maligantmelanoma cell line C24e32 (EP-A-0 493686), KHm-3/P [J. Natl. CancerInst., 59, 775 (1977)] or G361 (ATCC CRL1424). The suspension was placedin a microtube (Tref) and centrifuged (3,000 rpm, 2 minutes) to wash thecells, 50 μl of KM966 or KM967 (50 μg/ml) was added, the mixture wasstirred, and the reaction was allowed to proceed at 4° C. for 1 hour.Then, the cells were washed three times by centrifugation with PBS, 20μl of fluorescein isocyanate-labeled protein A (30-fold dilution;Boehringer Mannheim Yamanouchi) was added and, after stirring, thereaction was allowed to proceed at 4° C. for 1 hour. Then, the cellswere washed three times by centrifugation with PBS, then suspended inPBS and subjected to analysis using flow cytometer EPICS Elite(Coulter). In a control run, the same procedure as described above wasfollowed without adding the chimeric human antibody. The results thusobtained are shown in Table 2. The chimeric human antibody KM966 reactedwith 9 (NCI-H128, SBC-1, SBC-3, SBC-5, Lu-139, Lu-130, Lu-135, Lu-134-Band Lu-140) of the 14 lung small cell carcinoma cell lines, 2 (PC-10 andCalu-1) of the 5 lung squamous carcinoma cell lines, 2 (PC-9 andRERF-LC-MS) of the 5 lung adenocarcinoma cell lines, 2 (PC-13 andSK-LC-6) of the 4 lung large cell carcinoma cell lines, 7 (YT-nu, NAGAI,NB-1, IMR32, GOTO, NB-9 and SK-N-MC) of the 7 neuroblastoma cell linesand 4 (SK-MG-4, A172, T98G and U-118MG) of the 4 brain tumor (glioma)cell lines. On the other hand, the chimeric human antibody KM967 did notreact with any of the cultured cell lines. The above results indicatethat the chimeric human antibody KM966 is useful in the diagnosis andtreatment of brain tumors, peripheral nervous system tumors and lungcancer, among others.

                  TABLE 2                                                         ______________________________________                                                         KM966 (%)    KM967 (%)                                       Cell line        (50 μg/ml)                                                                              (50 μg/ml)                                   ______________________________________                                        Lung small cell carcinoma                                                                      9/14    (64)     0/14  (0)                                   Lung squamous cell                                                                            2/5      (40)    0/5    (0)                                   carcinoma                                                                     Lung adenocarcinoma                                                                           2/5      (40)    0/5    (0)                                   Lung large cell carcinoma                                                                     2/4      (50)    0/4    (0)                                   Neuroblastoma   7/7      (100)   0/7    (0)                                   Brain tumor (glioma)                                                                          4/4      (100)   0/4    (0)                                   Leukemia         0/14     (0)     0/14  (0)                                   Malignant melanoma                                                                            0/3       (0)    0/3    (0)                                   ______________________________________                                    

15. In vitro Antitumor Activity of the Chimeric Human Anti-GM₂ AntibodyKM966: Complement Dependent Cytotoxicity (CDC)

(1) Preparation of Target Cells

The target cells SBC-3, Lu-135, PC-10, RERF-LC-MS, PC-13, NAGAI, GOTO orA172, cultured in RPMI1640 medium supplemented with 10% FCS, wereadjusted to a cell concentration of 5×10⁶ cells/ml, Na₂ ⁵¹ CrO₄ wasadded to a concentration of 100 μCi/5×10⁶ cells, then the reaction wasallowed to proceed at 37° C. for 1 hours, and the cells were washedthree times with the medium. The cells were then allowed to stand in themedium at 4° C. for 30 minutes for spontaneous dissociation and then,after centrifugation, the medium was added to adjust the cellconcentration to 1×10⁶ cells/ml.

(2) Preparation of the Complement

Sera from three healthy subjects were combined and used as a complementsource.

(3) CDC Activity Measurement

The chimeric human anti-GM₂ antibody KM966 or mouse anti-GM₂ antibodyKM696 (FERM BP-3337) was added to wells of 96-well U-bottom plateswithin the final concentration range of 0.5 to 50 μg/ml and then 5×10⁴cells/well of the target cells prepared in (1) were added. The reactionwas allowed to proceed at room temperature for 30 minutes. Aftercentrifugation, the supernatants were discarded, 150 μl of the humanserum obtained in (2) was added to each well (final concentration 15%v/v), and the reaction was allowed to proceed at 37° C. for 1 hour.After centrifugation, the amount of ⁵¹ Cr in each supernatant wasdetermined using a gamma counter. The amount of spontaneouslydissociated ⁵¹ Cr was determined by adding to the target cells themedium alone in lieu of the antibody and complement solutions andmeasuring the amount of ⁵¹ Cr in the supernatant in the same manner asmentioned above. The total amount of dissociated ⁵¹ Cr was determined byadding to the target cells 5 N sodium hydroxide in lieu of the antibodyand complement solutions and measuring the amount of ⁵¹ Cr in thesupernatant in the same manner as mentioned above. The CDC activity wascalculated as follows: ##EQU1##

The results thus obtained are shown in FIGS. 20 to 23. It was shown thatthe chimeric human antibody KM966 show CDC activity against all thecells tested.

16. In vitro Antitumor Activity of the Chimeric Human Anti-GM₂ AntibodyKM966: Antibody Dependent Cell Mediated Cytotoxicity (ADCC)

(1) Preparation of Target Cells

The target cells SBC-3, Lu-135, PC-10, RERF-LC-MS, PC-13, NAGAI, GOTO orA172, cultured in RPMI1640 medium supplemented with 10% FCS, wereadjusted to a cell concentration of 1×10⁶ cells/ml, Na₂ ⁵¹ CrO₄ wasadded to a concentration of 50 μCi/1×10⁶ cells, then the reaction wasallowed to proceed at 37° C. for 1 hour, and the cells were washed threetimes with the medium. The cells were then allowed to stand in themedium at 4° C. for 30 minutes for spontaneous dissociation and then,after centrifugation, the medium was added to adjust the cellconcentration to 2×10⁵ cells/ml.

(2) Preparation of Effector Cells

Human venous blood (25 ml) was collected, 0.5 ml of heparin sodium(Takeda Chemical Industries; 1,000 units/ml) was added, and the mixturewas gently stirred. This mixture was centrifuged (1,500 to 1,800 g, 30minutes) using Polymorphprep (Nycomed Pharma AS), the lymphocyte layerwas separated and washed three times by centrifugation with RPMI-1640medium (15,00 to 1,800 g, 15 minutes), and the cells were suspended inRPMI1640 medium supplemented with 10% FCS (5×10⁶ cells/ml) for use aseffector cells.

(3) ADCC Activity Measurement

The chimeric human anti-GM₂ antibody KM966 or mouse anti-GM₂ antibodyKM696 were added to wells of 96-well U-bottom plates within the finalconcentration range of 0.05 to 5 μg/ml and then 50 μl (1×10⁴ cells/well)of the target cell suspension prepared in (1) and 100 μl (5×10⁵cells/well) of the effector cell suspension prepared in (2) were addedto each well (the ratio between the effector cells and target cellsbeing 50:1). The reaction was allowed to proceed at 37° C. for 4 hoursand, after centrifugation, the amount of ⁵¹ Cr in each supernatant wasmeasured using a gamma counter. The amount of spontaneously dissociated⁵¹ Cr was determined by adding to the target cells the medium alone inlieu of the antibody and effector cells and measuring the amount of ⁵¹Cr in the supernatant in the same manner as mentioned above. The totalamount of dissociated ⁵¹ Cr was determined by adding to the target cells5 N sodium hydroxide in lieu of the antibody and effector cells andmeasuring the amount of ⁵¹ Cr in the supernatant in the same manner asmentioned above. The ADCC activity was calculated as follows: ##EQU2##

The results thus obtained are shown in FIGS. 24 to 27. The chimericantibody KM966 showed ADCC activity against all the cells whereas themouse anti-GM₂ antibody KM696 showed no or low ADCC activity. The aboveresults indicate that the chimeric human antibody KM966 is moreeffective in the treatment of human cancer than the mouse antibodyKM-696.

REFERENCE EXAMPLE 1 Construction of the Vector pChiIgLA1 for ChimericHuman Antibody L Chain Expression

1. Isolation of the KM50 Cell-Derived Immunoglobulin H Chain Promoterand Enhancer Genes

(1) Preparation of Chromosomal DNAs from KM50 Cells, P3U1 Cells and RatKidney

Chromosal DNAs were prepared by the conventional method [Maniatis et al.(ed.): Molecular Cloning, 1989, p. 9.16], as follows.

KM50 cells (1.2×10⁸ cells), P3U1 cells (ATCC CRL1597) (2×10⁸ cells) anda rat kidney sample (frozen at -80° C. and then smashed to a sufficientextent using a wooden hammer) (1.6 g) were suspended in 2 ml of 10 mMTris-hydrochloride buffer (pH 7.5) containing 150 mM sodium chloride and10 mM ethylenediaminetetraacetic acid disodium salt (hereinafter,"EDTA"), 0.8 mg of proteinase K (Sigma) and 10 mg of sodium laurylsulfate (hereinafter, "SDS"), were added to each suspension, and thesuspension was incubated at 37° C. for 10 hours. Then, each mixture wasextracted once with an equal volume of phenol, twice with an equalvolume of chloroform and then once with an equal volume of ether, anddialyzed for 10 hours against 10 mM Tris-hydrochloride buffer (pH 7.5)containing 1 mM EDTA. The DNA solution was recovered from the dialysistube and ribonuclease A (Sigma) was added to the solution to a finalconcentration of 20 μg/ml. Each resultant solution was incubated at 37°C. for 6 hours for sufficient decomposition of RNA, 15 mg of SDS and 1mg of proteinase K were then added and the mixture was incubated at 37°C. for 10 hours. The mixture was then extracted twice with an equalvolume of phenol, twice with an equal volume of chloroform and twicewith an equal volume of ether and then dialyzed for 10 hours against 10mM Tris-hydrochloride buffer (pH 7.5) containing 1 mM EDTA. The DNAsolution was recovered from the dialysis tube for use as a chromosomalDNA sample. DNA concentration measurement in terms of the absorbance at260 nm revealed that the yield of chromosomal DNA from 1.2×10⁸ KM50cells was 1.6 mg, that from 2×10⁸ P3U1 cells 1.5 mg, and that from 1.6 gof rat liver 1.9 mg.

(2) Identification of the Active-Form Immunoglobulin H Chain Gene inKM50 Cells by Southern Blotting

The KM50 cell, p3U1 cell and rat kidney chromosomal DNAs obtained in (1)(3 μg each) were dissolved in 25 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride,15 units of XbaI (Takara Shuzo; hereinafter the restriction enzymes usedwere products of Takara Shuzo) was added and incubation was carried outat 37° C. for 2 hours for effecting cleavage at the XbaI sites. Eachreaction mixture was subjected to agarose gel electrophoresis, then DNAtransfer onto a nitrocellulose filter was effected by the method ofSouthern et al. [J. Mol. Biol., 98, 503 (1975)] and hybridization wascarried out by the conventional method [Kameyama et al.: FEBS Letters,244, 301-306 (1989)] using the mouse JH probe described in thelast-cited reference. The KM50 cell DNA alone gave a band at a sitecorresponding to about 9.3 kb. Therefore, the immunoglobulin XbaIfragment DNA was considered to code for the active-form immunoglobulin Hchain gene in KM50 cells.

(3) Construction of a KM50 Cell Genomic DNA Library

A 60-μg portion of the KM50 cell-derived chromosomal DNA obtained in (1)was dissolved in 250 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 100 mM sodium chloride, 150 unitsof XbaI was added, and incubation was conducted at 37° C. for 2 hoursfor causing cleavage at the XbaI sites. The reaction mixture wasfractionated by agarose gel electrophoresis and a KM50 cell-derived 9.3kb DNA fraction sample (about 2 μg) was recovered using, for example,the DEAE paper method [Maniatis et al. (ed.): Molecular Cloning, 1989,p. 6.24]. Separately, 3 μg of Lambda ZAP (Stratagene), for use as thevector, was dissolved in 200 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 50units of XbaI was added, and the mixture was incubated at 37° C. for 2hours to effect cleavage at the XbaI sites. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, whereby about 3 μg of DNA was recovered. This DNA wasdissolved in 100 μl of 100 mM Tris-hydrochloride buffer (pH 7.5), 1 unitof alkaline phosphatase (Takara Shuzo) was added, dephosphorylation waseffected at the restriction enzyme cleavage ends of the vector DNA. Thereaction mixture was subjected to phenol-chloroform extraction and thento ethanol precipitation, whereby 2 μg of DNA was recovered. This DNAwas dissolved in 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 1 mM EDTA for use as a vector sample. Two tenths μg of thevector DNA sample and 0.2 μg of the KM50 cell-derived 9.3 kb DNA samplewere dissolved in 5 μl of T4 ligase buffer, 175 units of T4 ligase(Takara Shuzo) was added, and the mixture was incubated at 4° C. for 3days. A 2-μl portion of this mixture was packaged into the lambda phageby the conventional method [Maniatis et al. (ed.): Molecular Cloning,1989, p. 2.95] using Giga Pak Gold (Stratagene), and the packagingmixture was used to transfect Escherichia coli BB4 to give 200,000 phageclones. Among them, 100,000 clones were fixed on a nitrocellulose filterby the conventional method [Maniatis et al. (ed.): Molecular Cloning,1989, p. 2.112].

(4) Selection of a Recombinant DNA Containing the Gene for the H ChainVariable Region of an Immunoglobulin Occurring as an Active Form in KM50Cells (Anti-Human Serum Albumin)

From among the phage library composed of 100,000 clones, as constructedin (3), two clones firmly associable at 65° C. with the ³² P- labeledmouse JH probe [labeled by the method of Kameyama et al. [FEBS Letters,44, 301-306 (1989)]] were isolated. The phage DNA was recovered fromthem by the conventional method [Maniatis et al. (ed.): MolecularCloning, 1989, p. 2.118-2.169], whereupon the 9.3 kb XbaI fragment ofthe KM50 cell-derived chromosomal DNA was found to have been insertedtherein.

(5) Base Sequence of the Gene for the H Chain Variable Region of theImmunoglobulin Occurring as an Active Form in KM50 Cells (Anti-HumanSerum Albumin)

For the two clones obtained in (4), restriction enzyme cleavage mapswere prepared by conducting digestion using various restriction enzymes,whereby it was revealed that the same DNA fragment (9.3 kb) had beeninserted therein (FIG. 28). Therefore, those portions of this 9.3 kb DNAfragment which were supposed to be coding for the rat immunoglobulin Hchain promoter region and variable region were sequenced by the methodof Sanger [Sanger et al.: Proc. Natl. Acad. Sci. U.S.A., 74, 5463(1977); AMERSHAM M13 cloning and sequencing handbook]. In SEQ ID NO:16,the portion containing the octamer sequence such as ATGCAAAT and theTATA box sequence such as TTGAAAA is considered to be the immunoglobulinpromoter region.

2. Construction of Heterologous Protein Expression Vectors Using thePromoter and Enhancer for the H Chain Variable Region Gene for anImmunoglobulin Occurring as an Active form in KM50 Cells (Anti-HumanSerum Albumin)

(1) Construction of pKMB11

A 1-μg portion of the 9.3 kb immunoglobulin H chain variable region genefragment obtained in Paragraph 1 (5) was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units each of BglII and HindIII wereadded, and the mixture was incubated at 37° C. for 2 hours for causingcleavage at the BqlII and HindIII sites. The reaction mixture wassubjected to agarose gel electrophoresis and 0.01 μg of a DNA fragmentcontaining the 0.8 kb immunoglobulin promoter was recovered. Then, 1 μgof the plasmid pBR322-BglII [Kuwana et al.: FEBS Letters, 219, 360(1987)] was dissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10units of BlII and 10 units of HindIII were added, and the mixture wasincubated at 37° C. for 2 hours to effect cleavage at the BglII andHindIII sites. The reaction mixture was subjected to agarose gelelectrophoresis and a DNA fragment of about 4.2 kb in size wasrecovered. The thus-obtained pBR322-BglII-derived DNA fragment (about4.2 kb, 0.1 μg) and immunoglobulin promoter-containing DNA fragment(0.01 μg) were dissolved in 20 μl of T4 ligase buffer, 175 units of T4DNA ligase (Takara Shuzo) was added, and the mixture was incubated at 4°C. for 1 day. The reaction mixture was used to transform Escherichiacoli HB101 [J. Mol. Biol., 41, 459 (1969)] by the method of Scott et al.[Masaru Shigesada: Saibo Kokagu (Cell Engineering), 2, 616 (1983)] togive an Ap-resistant colony. The recombinant plasmid DNA was recoveredfrom this colony. Plasmid pKMB11, shown in FIG. 29, was thus obtained.

(2) Construction of pKMD6

For providing an appropriate restriction enzyme site downstream from theimmunoglobulin promoter, the plasmid pKMB11 constructed in (1) wasdigested at the NcoI site using the nuclease BAL31. Thus, 10 μg of theplasmid pKMB11 was dissolved in 100 μl of 10 mM Tris-hydrochloridebuffer (pH 7.5) containing 6 mM magnesium chloride and 50 mM potassiumchloride, 30 units of NcoI was added, and the mixture was incubated at37° C. for 2 hours to effect cleavage at the NcoI site. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation, the whole amount of the DNA fragment wasdissolved in 100 μl of BAL31 buffer [20 mM Tris-hydrochloride buffer (pH8.0) containing 600 mM sodium chloride, 12 mM calcium chloride, 12 mMmagnesium chloride and 1 mM EDTA], 0.25 unit of BAL31 [Bethesda ResearchLaboratories (BRL)]] was added, and digestion was carried out at 37° C.for 5 seconds. The reaction was terminated by extraction with phenol andsubjected to chloroform extraction and then to ethanol precipitation,and 1 μg of DNA was recovered. A 0.1-μg portion of this DNA and 0.01 μgof a synthetic DNA linker (SalI) were dissolved in 20 μl of T4 ligasebuffer, 175 units of T4 DNA ligase was added, and the mixture wasincubated at 4° C. for 1 day. The reaction mixture was used to transformEscherichia coli HB101 by the method of Scott et al. An Ap-resistantcolony was obtained and the recombinant plasmid DNA was recovered fromthis colony to give the plasmid pKMD6 shown in FIG. 30. For thisplasmid, the portion of BAL31 digestion was sequenced by the method ofSanger, whereupon deletion was found to the third base (303rd base inSEQ ID NO:16) toward the upstream of the initiation codon ATG forimmunoglobulin.

(3) Construction of pEPKMA1, pEPKMB1 and pAGE501

The original immunoglobulin promoter and enhancer are positionallyseparated. Therefore, it was necessary to construct a vector containingthe promoter and enhancer connected to each other for use of said vectoras a heterologous protein expression vector. Accordingly, the followingprocedure was followed.

Thus, 1 μg of the 9.3 kb immunoglobulin H chain variable region geneobtained in Paragraph 1 (5) was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of EcoRV and 10 units of XbaI wereadded, and the mixture was incubated at 37° C. for 2 hours for causingcleavage at the EcoRV and XbaI sites. The reaction mixture was subjectedto agarose gel electrophoresis and 0.1 μg of a DNA fragment (about 1 kb)containing the immunoglobulin enhancer region was recovered. Separately,1 μg of the plasmid pKMD6 obtained in (2) was dissolved in 100 μl of 10mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of BglII was added, and the mixturewas incubated at 37° C. for 2 hours to effect cleavage at the BglIIsite. After phenol-chloroform extraction, the DNA was precipitated withethanol and dissolved in a total of 40 μl of DNA polymerase I buffer, 6units of Escherichia coli-derived DNA polymerase I Klenow fragment wasadded, and the reaction was allowed to proceed at 16° C. for 90 minutesfor rendering the 5' protruding ends formed upon BglII digestionblunt-ended. The reaction was terminated by extraction with phenol, themixture was extracted with chloroform and then subjected to ethanolprecipitation, the DNA obtained was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of HindIII was added, and themixture was incubated at 37° C. for 2 hours to effect cleavage at theHindIII site. The reaction mixture was subjected to agarose gelelectrophoresis and 0.1 μg of a DNA fragment (about 0.8 kb) containingthe immunoglobulin promoter region was recovered. Then, 0.2 μg of theplasmid pUC18 [Messing: Methods in enzymology 101, 20 (1983)] wasdissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 unitsof HindIII and 10 units of XbaI were added, and the mixture wasincubated at 37° C. for 2 hours for causing cleavage at the HindIII andXbaI sites. The reaction mixture was subjected agarose gelelectrophoresis and 0.1 μg of a DNA fragment of about 2.7 kb in size wasrecovered. The thus-obtained pPKMD6-derived 0.8 kb DNA fragment (0.1μg), immunoglobulin enhancer region-containing DNA fragment (0.02 μg)and pUC18 (0.1 μg) were dissolved in 20 μl of T4 ligase buffer, 175units of T4 DNA ligase was added, and the mixture was incubated at 4° Cfor 1 day. The reaction mixture was used to transform Escherichia coliHB101 to give an Ap-resistant colony. The recombinant plasmid DNA wasrecovered from this colony to give pEPKMA1 shown in FIG. 31.

Then, 1 μg of the plasmid pEPKMAl was dissolved in 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of XbaI was added, and the mixturewas incubated at 37° C. for 2 hours for causing cleavage at the XbaIsite. After phenol-chloroform extraction, the resultant DNA fragment wasprecipitated with ethanol and dissolved in a total of 40 μl of DNApolymerase I buffer, 6 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was allowed to proceed at16° C. for 90 minutes for rendering the cohesive ends formed upon XbaIdigestion blunt-ended. The reaction was terminated by extraction withphenol and, after chloroform extraction, the DNA fragment was recoveredby ethanol precipitation. This DNA fragment and a synthetic DNA linkerXhoI (Takara Shuzo) (0.01 μg) were dissolved in 20 μl of T4 ligasebuffer, 175 units of T4 DNA ligase was added, and the mixture wasincubated at 4° C. for 1 day. The reaction mixture was used to transformEscherichia coli HB101 to give an Ap-resistant colony. The recombinantplasmid DNA was recovered from this colony to give pEPKMB1 shown in FIG.32.

Then, the SV40 early gene promoter and enhancer regions (hereinafterabbreviated as P_(SE)) of the heterologous gene expression vectorpAGE107 for use in animals [Miyaji et al.: Cytotechnology, 3, 133-140(1990)] were replaced with the KM50-derived immunoglobulin H chainpromoter and enhancer (hereinafter abbreviated as P_(IH)) of pEPKMB1 inthe following manner.

One μg of the plasmid pAGE107 was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 150 mM sodium chloride, 10 units of SalI and 10 units of XhoI wereadded, and the mixture was incubated at 37° C. for 2 hours to effectcleavage at the SalI and XhoI sites. The reaction mixture was subjectedto agarose gel electrophoresis and 0.5 μg of a DNA fragment (about 5.95kb) containing the G418 resistance gene, among others, was recovered.Then, 1 μg of the plasmid pEPKMB1 was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 150 mM sodium chloride, 10 units of SalI and 10 units of XhoI wereadded, and the mixture was incubated at 37° C. for 2 hours to effectcleavage at the SalI and XhoI sites. The reaction mixture was subjectedto agarose gel electrophoresis and 0.1 μg of a DNA fragment (about 1.7kb) containing the immunoglobulin promoter and enhancer regions wasrecovered. The thus-obtained pAGE107-derived 5.95 kb DNA fragment (0.1μg) and immunoglobulin promoter and enhancer region-containing DNAfragment (0.02 μg) were dissolved in 20 μl of T4 ligase buffer, 175units of T4 DNA ligase was added, and the mixture was incubated at 4° C.for 1 day. The reaction mixture was used to transform Escherichia coliHB101. An Ap-resistant colony was isolated and the recombinant plasmidDNA was recovered therefrom to give pAGE501 shown in FIG. 33.

(4) Construction of pAGE109

A plasmid, pAGE109, derived from pAGE106 by deletion of one of the twoEcoRI sites in pAGE106 was constructed as follows.

Thus, 2 μg of the heterologous gene expression vector pAGE106 for use inanimal cells as described in EP-A-0 405 285 was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride; 10 units each of EcoRI and SacI were furtheradded, and digestion was conducted at 37° C. for 4 hours. The reactionmixture was fractionated by agarose gel electrophoresis and about 1.5 μgof a DNA fragment (4.3 kb) resulting from cleavage of pAGE106 with EcoRIand SacI and containing the SV40 early gene promoter and G418 resistancegene was recovered. Then, this DNA fragment was dissolved in a total of40 μl of DNA polymerase I buffer, 5 units of Escherichia coli-derivedDNA polymerase I large fragment was added, and the reaction wasconducted at 16° C. for 2 hours for rendering the 3' protruding endsformed upon SalI digestion and the 5' protruding ends formed upon EcoRIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was dissolved in 20 μl of T4 ligase buffer; 350 units of T4DNA ligase was further added to the mixed solution, and ligation wascarried out at 4° C. for 4 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 to give the plasmidpAGE109 shown in FIG. 34.

(5) Construction of pAGE502

For replacing the SV40 promoter and enhancer of pAGE107 with theimmunoglobulin H chain promoter and enhancer, a plasmid named pAGE502was constructed as follows.

Two μg of pAGE107 described in EP-A-0 405 285 was added to 100 μl of 10mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of HindIII was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon HindIIIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10units of XhoI was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.5 μg of a DNA fragment (about 5.95 kb),resulting from cleavage of pAGE107 with XhoI and HindIII and containingthe G418 resistance gene and Ap resistance, was recovered.

Two μg of pAGE501 obtained in (3) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 175 mM sodium chloride, 10 units of SalI was further added, anddigestion was carried out at 37° C. for 4 hours. The reaction mixturewas subjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon SalIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10units of XhoI was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.2 μg of a DNA fragment (1.8 kb) resultingfrom cleavage of pAGE501 with XhoI and SalI and containing the KM50 cellimmunoglobulin H chain promoter and enhancer was recovered.

Then, 0.1 μg of the HindIII-XhoI fragment (about 5.95 kb) of pAGE107 asobtained above and 0.1 μg of the SalI-XhoI fragment (about 1.8 kb) ofpAGE501 were dissolved in a total of 20 μl of T4 ligase buffer; 350units of T4 DNA ligase was added to the solution, and the mixture wasincubated at 4° C. for 1 day. The thus-obtained recombinant plasmid DNAwas used to transform Escherichia coli HB101 to give the plasmid pAGE502shown in FIG. 35.

(6) Construction of pAGE503

A plasmid named pAGE503 derived from pAGE502 by deletion of one of thetwo EcoRI sites was constructed as follows.

Two μg of pAGE109 obtained in (4) was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride; 10 units of HindIII and 10 units of ClaI werefurther added, and digestion was carried out at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.2 μg of a DNA fragment (about 1 kb) resulting from cleavage ofpAGE109 with ClaI and HindIII and containing the poly-A signal gene forthe beta globulin and SV40 early genes was recovered.

Then, 2 μg of pAGE502 obtained in (5) was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of HindIII and 10 units of ClaI werefurther added, and digestion was conducted at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 1 μg of a DNA fragment (about 6.1 kb) resulting from cleavage ofpAGE502 with HindIII and ClaI and containing the KM50 cellimmunoglobulin H chain promoter and enhancer genes, the Ap resistancegene and the G418 resistance gene was recovered by the DEAE papermethod. Then, 0.1 μg of the HindIII-ClaI fragment (about 1 kb) ofpAGE109 as obtained above and 0.1 μg of the HindIII-ClaI fragment (about6.1 kb) of pAGE502 as obtained above were dissolved in a total of 20 μlof T4 ligase buffer, 350 units of T4 DNA ligase was added to thesolution, and the mixture was incubated at 4° C. for 1 day. Thethus-obtained recombinant plasmid DNA was used to transform Escherichiacoli HB101 and the plasmid pAGE503 shown in FIG. 36 was obtained.

(7) Construction of pSE1d1

A plasmid named pSE1d1 was constructed by introducing the dhfr gene intopAGE107, as follows.

Two μg of pAGE107 described in EP-A-0 405 825 was added to 100 μl of 100mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of EcoRI was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon EcoRIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 50 mM sodium chloride; 10units of HindIII was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.5 μg of a DNA fragment (about 5.6 kb)resulting from cleavage of pAGE107 with EcoRI and HindIII and containingthe G418 resistance gene and Ap resistance gene was recovered.

Two μg of pSV2-dhfr [Subramani et al.: Mol. Cell. Biol., 1, 854 (1981)]was added to 100 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 unitsof BglII was further added, and digestion was carried out at 37° C. for4 hours. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation, the precipitate wasdissolved in a total of 40 μl of DNA polymerase I buffer, 5 units ofEscherichia coli-derived DNA polymerase I Klenow fragment was added, andthe reaction was conducted at 16° C. for 2 hours for rendering the 5'protruding ends formed upon BglII digestion blunt-ended. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation, the precipitate was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of HindIII was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.2 μg of apSV2-dhfr DNA fragment (0.76 kb) resulting from cleavage with BglII andHindIII and containing the dehydrofolate reductase (dhfr) gene wasrecovered.

Then, 0.1 μg of the HindIII-EcoRI fragment (about 5.6 kb) of PAGE107, asobtained above, and 0.1 μg of the BglII-HindIII fragment (about 0.76 kb)of pSV2-dhfr, as obtained above, were dissolved in a total of 20 μl ofT4 ligase buffer; 350 units of T4 DNA ligase was added to the solution,and the mixture was incubated at 4° C. for 1 day. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe plasmid pSE1d1 shown in FIG. 37 was obtained.

(8) Construction of pSE1d2

A plasmid named pSE1d2 was constructed by deleting the HindIII cleavagesite from pSE1d1, as follows.

Thus, 2 μg of pSE1d1 obtained in (7) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of HindIII was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon HindIIIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was dissolved in 20 μl of T4 ligase buffer, 350 units of T4DNA ligase was added to the solution, and the mixture was incubated at4° C. for 1 day. The thus-obtained recombinant plasmid DNA was used totransform Escherichia coli HB101 and the plasmid pSE1d2 shown in FIG. 38was obtained.

(9) Construction of pIg1SE1d2

A plasmid named pIg1SE1d2 was constructed by introducing the dhfr geneinto pAGE503, as follows.

Two μg of pAGE503 obtained in (6) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of ClaI was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon ClaIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 50 mM sodium chloride; 10units of MluI was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1 μg of a DNA fragment (about 5.4 kb)resulting from cleavage of pAGE503 with ClaI and MluI and containing theKM50 immunoglobulin H chain promoter and enhancer was recovered.

Then, 2 μg of pSE1d2 obtained in (8) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of XhoI was further added, anddigestion was carried out at 37° C. for 4 hours. The reaction mixturewas subjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon XhoIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10units of MluI was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1 μg of a DNA fragment (about 3.8 kb)resulting from cleavage of pSE1d2 with XhoI and MluI and containing thedhfr gene was recovered.

Then, 1 μg of the ClaI-MluI fragment (about 5.4 kb) of pAGE503 asobtained above and 1 μg of the XhoI-MluI fragment (about 3.8 kb) ofpSE1d2 as obtained above were dissolved in a total of 20 μl of T4 ligasebuffer, 350 units of T4 DNA ligase was added to the solution, and themixture was incubated at 4° C. for 1 day. The thus-obtained recombinantplasmid DNA was used to transform Escherichia coli HB101 and the plasmidpIg1SE1d2 shown in FIG. 39 was obtained.

(10) Construction of pIg1SE1d3

A plasmid named pIg1SE1d3 was constructed by deleting the ApaI cleavagesite from pIg1SE1d2, as follows.

Two μg of pIg1SE1d2 obtained in (9) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride,10 units of ApaI was further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation, the precipitate wasdissolved in a total of 40 μl of DNA polymerase I buffer, 5 units ofEscherichia coli-derived DNA polymerase I Klenow fragment was added, andthe reaction was carried out at 16° C. for 2 hours for rendering the 3'protruding ends formed upon ApaI digestion blunt-ended. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation, the precipitate was dissolved in 20 μl of T4ligase buffer, 350 units of T4 ligase was added to the solution, andligation was effected at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe plasmid pIg1SE1d3 shown in FIG. 40 was obtained.

(11) Construction of pIg1SE1d4

For providing pIg1SE1d3 with a cloning site between the HindIII cleavagesite and EcoRI cleavage site, a plasmid named pIg1SE1d4 was constructedcontaining the synthetic DNA defined by SEQ ID NO:17 as an insert, asfollows.

Two μg of pIg1SE1d3 obtained in (10) was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units each of HindIII and EcoRI werefurther added, and digestion was carried out at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 1 μg of a DNA fragment (about 9.2 kb) resulting from cleavage ofpIg1SE1d3 with HindIII and EcoRI and containing the KM50 cellimmunoglobulin H chain promoter, enhancer, Ap resistance gene, G418resistance gene and dhfr gene was recovered.

Then, 0.1 μg of the HindIII-EcoRI fragment (about 9.2 kb) of pIg1SE1d3as obtained above and 10 ng of the synthetic DNA (SEQ ID NO:17) were atotal of 20 μl of T4 ligase buffer, 350 units of T4 DNA ligase was addedto the solution, and the mixture was incubated at 4° C. for 1 day. Thethus-obtained recombinant plasmid DNA was used to transform Escherichiacoli HB101 and the plasmid pIg1SE1d4 shown in FIG. 41 was obtained.

3. Preparation of the Moloney Mouse Leukemia Virus Long Terminal Repeat(Hereinafter Abbreviated as "MoLTR")

It is known that MOLTR has promoter and enhancer activity [Kuwana etal.: Biochem. Biophys. Res. Commun., 149, 960 (1987)]. Therefore, forusing MOLTR as a promoter and enhancer in vectors for chimeric humanantibody expression, a plasmid, pPMOL3, containing MOLTR was constructedas follows.

Three μg of pPMOL1 described in JP-A-1-63394 was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 7 mM magnesium chlorideand 6 mM 2-mercaptoethanol, 10 units of ClaI was further added, anddigestion was carried out at 37° C. for 4 hours. The reaction mixturewas subjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was carried out at 16° C.for 2 hours for rendering the 5' protruding ends formed upon ClaIdigestion blunt-ended. The reaction was terminated by extraction withphenol, the reaction mixture was subjected to chloroform extraction andthen to ethanol precipitation, and 2 μg of a DNA fragment was recovered.This DNA fragment and 0.01 μg of a synthetic DNA linker XhoI (TakaraShuzo) were dissolved in 20 μl of T4 ligase buffer, 175 units of T4 DNAligase was added, and the mixture was incubated at 4° C. for 1 day. Thereaction mixture was used to transform Escherichia coli HB101 and theplasmid pPMOL2 shown in FIG. 42 was obtained. Then, 3 μg of pPMOL2 wasadded to 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 7mM magnesium chloride, 10 mM sodium chloride and 6 mM 2-mercaptoethanol,10 units of SmaI was further added, and digestion was conducted at 37°C. for 4 hours. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation, and 2 μg of a DNA fragmentwas recovered. This DNA fragment and 0.01 μg of a synthetic DNA linker(EcoRI; Takara Shuzo) were dissolved in 20 μl of T4 ligase buffer, 175units of T4 DNA ligase was added, and the mixture was incubated at 4° C.for 1 day. The reaction mixture was used to transform Escherichia coliHB101 and the plasmid pPMOL3 shown in FIG. 43 was obtained.

4. Cloning of the Human Immunoglobulin IgGl H Chain Constant Region(Cγ1) cDNA and L Chain Constant Region (C_(K)) cDNA

(1) Isolation of mRNA from the Chimeric Antibody Producer Cell LineSP2-PC Chimera-1

Using mRNA extraction kit Fast Track (product number K1593-02)manufactured by Invitrogen, mRNA (6.2 μg) was isolated from 1×10⁸ cellsof the chimeric antibody producer cell line SP2-PC Chimera-1 describedin FEBS Letters, 244, 301-306 (1989) and capable of producing a chimericantibody having anti-phosphorylcholine activity.

(2) Construction of an SP2-PC Chimera-1 cDNA Library and Cloning of theHuman Immunoglobulin H Chain Constant Region (Cγ1) cDNA and L chainconstant region (C_(K)) cDNA

Starting with 2 μg of the mRNA obtained in (1) and using cDNA SynthesisKit (product number 27-9260-01) manufactured by Pharmacia, EcoRI adapterjoining was performed, followed by phosphorylation. The cDNA solutionobtained was subjected to phenol-chloroform extraction and then toethanol precipitation, and 4 μg of cDNA was recovered. This cDNA wasdissolved in 20 μl of sterilized water and then fractionated by agarosegel electrophoresis, and about 0.3 μg each of two DNA fragments, about1.8 kb and about 1.0 kb in size, were recovered.

Then, 5 μg of the vector pUC18 was added to 100 μl of 100 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 50 units of EcoRI was further added, anddigestion was carried out at 37° C. for 4 hours for cleaving the pUC18DNA at the EcoRI site. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, andabout 3 μg of a DNA fragment resulting from cleavage of pUC18 at theEcoRI site thereof was recovered.

Then, 0.1 μg of the EcoRI fragment (about 2.7 kb) of pUC18 as obtainedabove and 0.1 μg each of the 1.8 kb and 1.0 kb cDNA fragments preparedfrom SP2-PC Chimera-1 cells were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 DNA ligase was added to the solution, andligation was effected at 4° C. for 24 hours.

The thus-obtained recombinant plasmid DNA was used to transformEscherichia coli LE392. About 3,000 colonies obtained were fixed onto anitrocellulose filter. From among the strains firmly bound at 65° C. toprobes prepared by labeling the human immunoglobulin constant regionchromosomal genes (IgG1 H chain constant region Cγl and L chain constantregion C_(K)) [Kameyama et al.: FEBS Letters, 244, 301 (1989)] with ³²P, a plasmid (pPCVHhCGI1) associable with Cγ1 and another (pPCVLhCK1)associable with C_(K) were isolated.

(3) Introduction of an EcoRV Site into the Human Ig_(K) chain ConstantRegion Gene

An EcoRV site was introduced into the human Ig_(K) chain constant regionat a site near the 5' end thereof by site-directed mutagenesis using akit (catalog number Q6210) manufactured by Promega. The plasmidpPCVLhCK1 (2 μg) was added to 30 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 6 mM magnesium chloride and 50 mM sodium chloride,10 units of EcoRI and 10 units of KpnI were further added, and digestionwas conducted at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.2 μg of a DNAfragment (about 0.8 kb) resulting from cleavage of pPCVLhCK1 with KpnIand EcoRI and containing the human immunoglobulin L chain constantregion gene was recovered.

Then, 2 μg of pSELECT1 (a kit manufactured by Promega; catalog numberQ6210) was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 50 mM sodium chloride, 10 unitseach of EcoRI and KpnI were further added, and digestion was carried outat 37° C. for 4 hours. The reaction mixture was fractionated by agarosegel electrophoresis and about 1 μg of a DNA fragment (about 5.7 kb)resulting from cleavage of pSELECT1 with EcoRI and KpnI was recovered.

Then, 0.1 μg of the EcoRI-KpnI fragment (about 0.8 kb) of pPCVLhCK1 asobtained above and 0.1 μg of the EcoRI-KpnI fragment (about 5.7 kb) ofpSELECT1 as obtained above were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 DNA ligase was added to the solution, andligation was effected at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli JM109 andthe plasmid pchCKA7 shown in FIG. 44 was obtained.

Then, using pchCKA7 and using the synthetic DNA defined by SEQ ID NO:18as a mutagenic primer, the sequence covering the 12th base to 14 basefrom the N terminus of the human immunoglobulin L chain constant region,namely ACC, was converted to GAT and thus an EcoRV site was introducedinto that site, to give a plasmid named pchCKB1 (FIG. 45).

Then, the EcoRV site of pchCKB1 was converted to a HindIII cleavage sitein the following manner.

Thus, 2 μg of the plasmid pchCKB1 was added to 10 μl of 100 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of EcoRI was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived polymerase IKlenow fragment was added, and the reaction was carried out at 37° C.for 30 minutes for rendering the 5' protruding ends formed upon EcoRIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then ethanol precipitation, theprecipitate was dissolved, together with 0.1 μg of a HindIII linker(Takara Shuzo), in 20 μl of T4 ligase buffer; 350 units of T4 ligase wasadded to the solution, and ligation was effected at 4° C. for 24 hours.The thus-obtained recombinant plasmid DNA was used to transformEscherichia coli HB101 and the plasmid pchCKC1 shown in FIG. 46 wasobtained.

5. Construction of Vectors for Chimeric Human Antibody H ChainExpression

(1) Construction of a Vector to be Used in Constructing Chimeric HumanAntibody H Chain Expression Vectors (Vector for Chimeric Human AntibodyH Chain Expression)

The plasmid pIg1SE1d4 obtained in Paragraph 2 (11) (2 μg) was added to30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mMmagnesium chloride and 100 mM sodium chloride, 10 units each of EcoRVand ApaI were further added, and digestion was effected at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.5 μg of a DNA fragment (about 9.2 kb)resulting from cleavage of pIg1SE1d4 with EcoRV and ApaI was recovered.

Then, 2 μg of pPCVHhCGI1 obtained in Paragraph 4 (2) was added to 30 μlof 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesiumchloride, 10 units of ApaI and 10 units of SmaI were further added, anddigestion was conducted at 37° C. for 1 hour. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.2 μg of a DNAfragment (about 1 kb) resulting from cleavage of pPCVHhCGI1 with ApaIand SmaI and containing the human immunoglobulin H chain constant regiongene was recovered.

Then, 0.1 μg of the EcoRV-ApaI fragment (about 9.2 kb) of pIg1SE1d4 asobtained above and 0.1 μg of the ApaI-SmaI fragment (about 1 kb) ofpPCVHhCGI1 as obtained above were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 DNA ligase was added to the solution, andligation was conducted at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe vector pCHiIgHB2 for chimeric human antibody H chain expression asshown in FIG. 47 was obtained.

(2) Construction of a Vector to be Used in Constructing Chimeric HumanAntibody L Chain Expression Vectors (Vector for Chimeric Human AntibodyL Chain Expression)

The plasmid pIg1SE1d4 obtained in Paragraph 2 (11) (2 μg) was added to30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mMmagnesium chloride and 100 mM sodium chloride, 10 units of EcoRV and 10units of HindIII were further added, and digestion was effected at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.5 μg of a DNA fragment (about 9.2 kb)resulting from cleavage of pIg1SE1d4 with EcoRV and HindIII wasrecovered.

Then, 2 μg of pckCKC1 obtained in Paragraph 4 (3) was added to 30 μl of10 mM Tris-hydrochloride (pH 7.5) containing 6 mM magnesium chloride and100 mM sodium chloride, 10 units of EcoRV and 10 units of HindIII werefurther added, and digestion was carried out at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.2 μg of a DNA fragment (about 0.6 kb) resulting from cleavage ofpPCVLhCK1 with EcoRV and HindIII and containing the human immunoglobulinL chain constant region gene was recovered.

Then, 0.1 μg of the EcoRV-HindIII fragment (about 9.2 kb) of pIg1SE1d4as obtained above and 0.1 μg of the EcoRV-HindIII fragment (about 0.6kb) of pchCKC1 as obtained above were dissolved in a total of 20 μl ofT4 ligase buffer, 350 units of T4 DNA ligase was added to the solution,and ligation was carried out at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe vector pChiIgLA1 for chimeric human antibody L chain expression asshown in FIG. 48 was obtained.

REFERENCE EXAMPLE 2 Construction of a Chimeric Human Antibody H ChainExpression Vector, pChi641HA1

1. Isolation of mRNA from Mouse Anti-GD₃ Monoclonal AntibodyKM-641-Producing Hybridoma Cells

Using mRNA extraction kit Fast Track (product number K1593-02)manufactured by Invitrogen, 34 μg of mRNA was isolated from 1×10⁸ mouseanti-GD₃ monoclonal antibody KM-641-producing hybridoma cells obtainableas in Reference Example 1.

2. Construction of a KM-641 H Chain cDNA Library and a KM-641 L ChaincDNA Library

Using 3 μg of the mRNA obtained in Paragraph 1 and using cDNA synthesiskit ZAP-cDNA Synthesis Kit (product number sc200400) manufactured byStratagene, cDNA having an EcoRI adapter at the 5' terminus and cDNAhaving an XhoI adapter at the 3' terminus were synthesized. About 6 μgof each cDNA was dissolved in 10 μl of sterilized water and fractionatedby agarose gel electrophoresis. In this way, about 0.1 μg of a cDNAfragment having a size of about 1.8 kb and corresponding to the H chainand a cDNA fragment having a size of about 1.0 kb and corresponding tothe L chain were recovered. Then, 0.1 μg of the cDNA fragment of about1.8 kb in size, 0.1 μg of the cDNA fragment of about 1.0 kb in size and1 μg of Uni-ZAP XR (Stratagene; derived from the Lambda ZAPII vector bycleavage with EcORI and XhoI, followed by treatment with calf intestinealkaline phosphatase), to be used as the vector, were dissolved in T4ligase buffer; 175 units of T4 DNA ligase was added, and the mixture wasincubated at 12° C. for 10 hours and further at room temperature for 2hours. A 4-μl portion of the reaction mixture was packaged into thelambda phage by the conventional method [Maniatis et al. (ed.):Molecular Cloning, 1989, p. 2.95] using Giga Pak Gold (Stratagene),followed by transfection of Escherichia coli PLK-F with the packagingmixture by the conventional method [Maniatis et al. (ed.): MolecularCloning, 1989, p. 2.95-107]. As an H chain cDNA library and as an Lchain cDNA library, about 10,000 phage clones were respectivelyobtained. The phages were then fixed onto nitrocellulose filters by theconventional method [Maniatis et al. (ed.): Molecular Cloning, 1989, p.2.112].

3. Cloning of the Monoclonal Antibody KM-641 H Chain and L Chain cDNAs

Using probes prepared by labeling a mouse Cγ1 gene (mouse immunoglobulinconstant region chromosomal gene)-containing EcoRI fragment (about 6.8kb) [Roeder et al.: Proc. Natl. Acad. Sci. U.S.A., 78, 474 (1981)] and amouse C_(K) gene-containing HindIII-BamHI fragment (about 3 kb) [Sakanoet al.: Nature, 280, 288 (1979)] with ³² P, one phage clone stronglyassociable with the former probe at 65° C. and one phage clone stronglyassociable with the latter probe at 65° C. were isolated from the Hchain cDNA library and L chain cDNA library constructed in Paragraph 2in accordance with the conventional method [Maniatis et al. (ed.):Molecular Cloning, 1989, p. 2.108]. Then, by converting the phage clonesto pBluescript plasmids using CDNA synthesis kit ZAP-cDNA Synthesis Kit(product number sc200400) manufactured by Stratagene, a KM-641 H chaincDNA-containing recombinant plasmid, pKM641HA3, and a KM-641 L chaincDNA-containing recombinant plasmid, pKM641LA2, were obtained. Cleavageof pKM641HA3 and pKM641LA2 with EcoRI and XhoI revealed that a cDNAfragment of about 1.6 kb and a cDNA fragment of about 0.9 kb had beeninserted therein, respectively (FIG. 49).

4. Base Sequences of the Immunoglobulin Variable Regions in the KM-641 HChain cDNA (pKM641HA3) and KM-641 L Chain cDNA (pKM641LA2)

The base sequences of the immunoglobulin regions in pKM641HA3 andpKM641LA2 obtained in Paragraph 3 were determined by the dideoxy method[Maniatis et al. (ed.): Molecular Cloning, 1989, p. 13.42] usingSequenase Version 2.0 DNA Sequencing Kit (United States BiochemicalCorporation). The results obtained are shown in SEQ ID NO:19 and SEQ IDNO:20. In pKM641LA2, a methionine codon, presumably the initiation codonATG, was found in the vicinity of the 5' terminus and the cDNA was aleader sequence-containing full-length one. In pKM641HA3, no methionineinitiation codon was found and the leader sequence was partly lacking.

5. Construction of a KM-641-Derived Chimeric Human Antibody H ChainExpression Vector

A chimeric human antibody H chain expression vector was constructed byjoining the H chain variable region gene obtained by cleaving theplasmid pKM641HA3 at the AluI site near the 5' terminus of the variableregion gene and at the StyI site near the 3' terminus of the variableregion gene to the vector for chimeric human antibody H chain expressionas obtained in Reference Example 1 using the synthetic DNAs defined bySEQ ID NO:21 and SEQ ID NO:22 (FIG. 50).

First, the DNA defined by SEQ ID NO:22 composed of the base sequencefrom the 3' terminus of the immunoglobulin H chain variable region inpKM641HA3 to the StyI cleavage site near said 3' terminus and the basesequence from the 5' terminus of the immunoglobulin H chain constantregion in pAGE28 to the ApaI cleavage site near said 5' terminus andhaving a StyI cleavage site and an ApaI cleavage site on the respectivetermini (cf. FIG. 50) was synthesized using a DNA synthesizer. Thissynthetic DNA was then introduced into the plasmid pKM641HA3 in thefollowing manner.

Three μg of pKM641HA3 was added to 30 μl of 50 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride, 50 mM sodiumchloride and 1 mM DTT, 10 units of EcoRI and 10 units of StyI werefurther added, and digestion was effected at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.3 μg of a 0.41 kb DNA fragment was recovered. Then, 3 μg ofpAGE28 [Mizukami et al.: J. Biochem., 101, 1307-1310 (1987)] was addedto 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 7 mMmagnesium chloride and 6 mM 2-mercapto-ethanol, 10 units of EcoRI and 10units of ApaI were further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 2 μg of a 2.45 kb DNA fragment was recovered.Then, 0.1 μg of the EcoRI-StyI fragment (about 0.41 kb) of pKM641HA3, asobtained above, 0.1 μg of the EcoRI-ApaI fragment (about 2.45 kb) ofpAGE28, as obtained above, and 0.3 μg of the synthetic DNA, defined bySEQ ID NO:22, were dissolved in a total of 20 μl of T4 ligase buffer;350 units of T4 ligase was added to the solution, and ligation wasconducted at 4° C. for 24 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 and the plasmidpKM641HE1 shown in FIG. 51 was obtained.

Since pKM641HE1 had no leader sequence, the following measure was takento supplement the deficit using the synthetic DNA defined by SEQ IDNO:21.

pKM641HE1 (3 μg) was added to 30 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 7 mM magnesium chloride and 6 mM 2-mercaptoethanol,10 units of EcoRI and 10 units of ApaI were further added, and digestionwas effected at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.4 μg of a DNAfragment of about 0.42 kb in size was recovered. The EcoRI-ApaI fragment(about 0.42 kb; 0.4 μg) of pKM641HE1 was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 7 mM magnesium chloride,50 mM sodium chloride and 6 mM 2-mercaptoethanol, 10 units of AluI wasfurther added, and digestion was effected at 37° C. for 4 hours. Thereaction mixture was subjected to phenol-chloroform extraction and thento ethanol precipitation, and about 0.3 μg of a DNA fragment of about0.4 kb in size was recovered.

Then, 0.1 μg of the AluI-ApaI fragment (about 0.4 kb) of pKM641HE1 asobtained above, 0.1 μg of the EcoRI-ApaI fragment (about 2.45 kb) ofpAGE28 as obtained above and 0.3 μg of the synthetic DNA defined by SEQID NO:21 were dissolved in a total of 20 μl of T4 ligase buffer; 350units of T4 ligase was added to the solution, and ligation was carriedout at 4° C. for 24 hours. The thus-obtained recombinant plasmid DNA wasused to transform Escherichia coli HB101 and the plasmid pKM641HF1 shownin FIG. 52 was obtained.

Then, the immunoglobulin H chain variable region of pKM641HF1 wasintroduced into the vector pChiIgHB2 for chimeric human antibody H chainexpression, as follows.

pKM641HF1 (3 μg) was added to 30 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 7 mM magnesium chloride and 6 mM 2-mercaptoethanol,10 units of EcoRI and 10 units of ApaI were further added, and digestionwas effected at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.5 μg of a 0.44kb DNA fragment was recovered. Then, 3 μg of pChiIgHB2 was added to 30μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 7 mM magnesiumchloride and 6 mM 2-mercaptoethanol, 10 units of EcoRI and 10 units ofApaI were further added, and digestion was conducted at 37° C. for 4hours. The reaction mixture was subjected to phenol-chloroformextraction and about 3 μg of DNA was recovered. Then, 0.1 μg of theEcoRI-ApaI fragment (about 0.44 kb) of pKM641HF1 as obtained above and0.1 μg of the EcoRI-ApaI fragment (about 10.1 kb) of pChiIgHB2 asobtained above were dissolved in a total of 20 μl of T4 ligase buffer;350 units of T4 ligase was added to the solution, and ligation wascarried out at 4° C. for 24 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 and the plasmidpChi641HA1 shown in FIG. 53 was obtained.

Then, the KM50-derived immunoglobulin H chain promoter and enhancerregion of pChi641HA1 was replaced with MoLTR, as follows.

pChi641HA1 (3 μg) was added to 30 μl of 50 mM Tris-hydrochloride buffer(pH 7.5) containing 10 mM magnesium chloride, 50 mM sodium chloride and1 mM DTT, 10 units of EcoRI and 10 units of XhoI were further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.2 μg of a DNAfragment of about 8.8 kb in size was recovered. pPMOL3 (3 μg) obtainedin Example 1, Paragraph 2 was added to 30 μl of 50 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride, 50 mM sodiumchloride and 1 mM DTT; 10 units of EcoRI and 10 units of XhoI werefurther added, and digestion was carried out at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.3 μg of a MoLTR-containing DNA fragment (0.63 μg) was recovered.Then, 0.1 μg of the EcoRI-XhoI fragment of pChi641HA1 and 0.1 μg of theEcoRI-XhoI fragment of pPMOL3 were dissolved in 20 μl of T4 ligasebuffer, 175 units of T4 DNA ligase was added, and the mixture wasincubated at 4° C. for 1 day. The reaction mixture was used to transformEscherichia coli HB101 and the KM-641-derived chimeric human H chainexpression vector pChi641HAM1 shown in FIG. 54 was obtained.

EXAMPLE 2 Production of Human CDR-Grafted Anti-GM₂ Antibodies (1)

1. Construction of DNAs Each Coding for Human CDR-Grafted Anti-GM₂Antibody H Chain Variable Region and Human CDR-Grafted Anti-GM₂ AntibodyL Chain Variable Region

(1) Construction of DNA Coding for Human CDR-Grafted Anti-GM₂ Antibody HChain Variable Region

A DNA coding for a human CDR-grafted anti-GM₂ antibody H chain variableregion, hKM796H, which contains amino acid sequences of SEQ ID NO:6, SEQID NO:7 and SEQ ID NO:8, was constructed in the following manner.

NEWM [BIO/TECHNOLOGY, 9, 266 (1991)] was used as human antibody H chainvariable region-encoding DNA to which each CDR was to be transplanted.DNAs set forth in SEQ ID NO:23 through NO:29 corresponding to NEWM inwhich each CDR was replaced with amino acid sequences of SEQ ID NO:6,SEQ ID NO:7 and SEQ ID NO:8 were synthesized using an automatic DNAsynthesizer (model 380A manufactured by Applied Biosystems Co., Ltd.).The thus-obtained synthetic DNAs (50 picomoles each) were dissolved in20 μl of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mMmagnesium chloride, 5 mM DTT, 0.1 mM EDTA and 0.5 mM ATP, 5 units of T4polynucleotide kinase was added, and 5'-phosphorylation was carried outat 37° C. for 30 minutes. Ten picomoles each of the resultingphosphorylated synthetic DNAs, which had restriction enzyme sites onboth ends, were ligated in the order of SEQ ID NO. (SEQ ID NO:23 throughNO:29) using a DNA ligation kit (Takara Shuzo) in accordance with themanufacturer's instruction attached to the kit to obtain a DNA, hKM796H,shown in FIG. 55. The amino acid sequence corresponding to hKM796H isshown in SEQ ID NO:36.

(2) Construction of DNA Coding for Human CDR-Grafted Anti-GM₂ Antibody LChain Variable Region

A DNA coding for a human CDR-grafted anti-GM₂ antibody L chain variableregion, hKM796L, which contains amino acid sequences of SEQ ID NO:9, SEQID NO:10 and SEQ ID NO:11, was constructed in the following manner.

REI [BIO/TECHNOLOGY, 9, 266 (1991)] was used as human antibody L chainvariable region-encoding DNA to which each CDR was to be transplanted.DNAs set forth in SEQ ID NO:30 through NO:35 corresponding to REI inwhich each CDR was replaced with amino acid sequences of SEQ ID NO:9,SEQ ID NO:10 and SEQ ID NO:11 were synthesized using an automatic DNAsynthesizer (model 380A manufactured by Applied Biosystems Co., Ltd.).The thus-obtained synthetic DNAs (50 picomoles each) were dissolved in20 μl of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mMmagnesium chloride, 5 mM DTT, 0.1 mM EDTA and 0.5 mM ATP, 5 units of T4polynucleotide kinase was added, and 5' -phosphorylation was carried outat 37° C. for 30 minutes. Ten picomoles each of the resultingphosphorylated synthetic DNAs, which had restriction enzyme sites onboth ends, were ligated in the order of SEQ ID NO. (SEQ ID NO:30 throughNO:35) using a DNA ligation kit (Takara Shuzo) in accordance with themanufacturer's instruction attached to the kit to obtain a DNA, hKM796L,shown in FIG. 56. The amino acid sequence corresponding to hKM796L isshown in SEQ ID NO:37.

2. Construction of Human CDR-Grafted Antibody H Chain Expression Vectorand Human CDR-Grafted Antibody L Chain Expression Vector

(1) Construction of Human CDR-Grafted Antibody H Chain Expression Vector

A NotI-ApaI fragment of the DNA coding for human CDR-grafted antibody Hchain variable region, obtained in Paragraph 1(1) of Example 2, wasligated to the plasmid pChi796HM1, obtained in Paragraph 7(3) of Example1, in the following manner (FIG. 57).

Three μg of pChi796HM1, obtained in Paragraph 7(3) of Example 1, weredissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of ApaI wereadded thereto and the mixture was allowed to react at 37° C. for 1 hour.The resulting mixture was subjected to ethanol precipitation and thethus-obtained precipitate was dissolved in 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Ten units of NotI were addedthereto to allow the mixture to react at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis to recover about2 μg of a DNA fragment of about 9.0 kb. Then, about 0.1 μg of thethus-obtained ApAI-NotI fragment of pChi796HM1 was ligated to 0.5 pmolesof the NotI-ApaI fragment of the DNA coding for human CDR-graftedantibody H chain variable region, obtained in Paragraph 1(1) of Example2, using a DNA ligation kit (Takara Shuzo). The resulting recombinantplasmid DNA was used to transform Escherichia coli HB101 and the plasmidphKM796HM1 shown in FIG. 57 was obtained.

Then, a human CDR-grafted antibody H chain expression vector wasconstructed by introducing β-globin 3' splicing signal into the plasmidphKM796HM1 in the following manner (FIG. 58).

Three μg of phKM796HM1 were added to 30 μl of 10 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT, 1 unitof KpnI was added thereto. The mixture was allowed to react at 37° C.for 10 minutes to effect partial digestion. The resulting mixture wassubjected to ethanol precipitation and the thus-obtained precipitate wasdissolved in 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 mMDTT. After adding 1 unit of XhoI, the mixture was allowed to react at37° C. for 10 minutes to effect partial digestion. The reaction mixturewas fractionated by agarose gel electrophoresis to recover about 0.2 μgof a DNA fragment of about 2.1 kb. Separately, 3 μg of pAGE148, obtainedin Paragraph 7(2) of Example 1, were added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT. Ten units of KpnI were added thereto to allow the mixtureto react at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation and the thus-obtained precipitate was dissolved in30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride, 100 mM sodium chloride and 1 mM DTT. After adding 10units of XhoI, the mixture was allowed to react at 37° C. for 1 hour andthen fractionated by agarose gel electrophoresis to recover about 1 μgof a DNA fragment of about 8.7 kb. One tenth μg of the thus-obtainedXhoI-KpnI fragment of phKM796HM1 was ligated to 0.1 μg of the XhoI-KonIfragment of pAGE148 using a DNA ligation kit (Takara Shuzo). Thethus-obtained recombinant plasmid DNA was used to transform Escherichiacoli HB101 to obtain the plasmid phKM796HMS1 shown in FIG. 58.

(2) Construction of Human CDR-Grafted Antibody L Chain Expression Vector

An EcoRI fragment having blunt ends of the DNA coding for humanCDR-grafted antibody L chain variable region, obtained in Paragraph 1(2) of Example 2, was ligated to the chimeric human antibody L chainexpression vector pChiIgLA1 in the following manner (FIG. 59).

Three μg of pChiIgLA1, obtained in Reference Example 1, were added to 30μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units ofEcoRI and 10 units of EcoRV were added thereto and the mixture wasallowed to react at 37° C. for 1 hour. The reaction mixture wasfractionated by agarose gel electrophoresis to recover about 1 μg of aDNA fragment of about 8.6 kb. Then, about 0.1 μg of the thus-obtainedEcoRI-EcoRV fragment of pChiIgLA1 was ligated to 0.5 pmoles of the EcoRIfragment having blunt ends derived from the DNA coding for humanCDR-grafted antibody L chain variable region, obtained in Paragraph 1(2)of Example 2, using a DNA ligation kit (Takara Shuzo). The resultingrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe plasmid phKM796LI1 shown in FIG. 59 was obtained.

Then, P_(MO) was introduced into the plasmid phKM796LI1 in the followingmanner (FIG. 60).

Three μg of phKM796LI1 were added to 30 μl of 50 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride, 100 mM sodiumchloride and 1 mM DTT, 10 units of EcoRI and 10 units of XhoI were addedthereto, and the mixture was allowed to react at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis torecover about 1 μg of a DNA fragment of about 8.2 kb. Separately, 3 μgof the chimeric human antibody H chain expression vector pChi641HAM1,obtained in Reference Example 2, were added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Ten units of EcoRI and 10 units ofXhoI were added thereto to allow the mixture to react at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis to recover about 0.3 μg of a DNA fragment of about 0.6kb. One tenth μg of the thus-obtained EcoRI-XhoI fragment of pChi641HAM1was ligated to 0.1 μg of the EcoRI-XhoI fragment of phKM796LI1 using aDNA ligation kit (Takara Shuzo). The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 to obtain the plasmidphKM796LM1 shown in FIG. 60.

Then, a human CDR-grafted antibody L chain expression vector wasconstructed by introducing β-globin 3' splicing signal into the plasmidphKM796LM1 in the following manner (FIG. 61).

Three μg of phKM796LM1 were added to 30 μl of 10 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10units of KpnI were added thereto, and the mixture was allowed to reactat 37° C. for 1 hour. The resulting mixture was subjected to ethanolprecipitation and the thus-obtained precipitate was dissolved in 30 μlof 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT. After adding 10 units ofXhoI, the mixture was allowed to react at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis torecover about 0.3 μg of a DNA fragment of about 1.6 kb. Separately, 3 μgof pAGE148, obtained in Paragraph 7(2) of Example 1, were added to 30 μlof 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride and 1 mM DTT. Ten units of KpnI were added thereto to allow themixture to react at 37° C. for 1 hour. The reaction mixture wassubjected to ethanol precipitation and the thus-obtained precipitate wasdissolved in 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 mMDTT. After adding 10 units of XhoI, the mixture was allowed to react at37° C. for 1 hour and then fractionated by agarose gel electrophoresisto recover about 1 μg of a DNA fragment of about 8.7 kb. One tenth μg ofthe thus-obtained XhoI-KpnI fragment of phKM796LM1 was ligated to 0.1 μgof the XhoI-KpnI fragment of pAGE148 using a DNA ligation kit (TakaraShuzo). The thus-obtained recombinant plasmid DNA was used to transformEscherichia coli HB101 to obtain the plasmid phKM796LMS1 shown in FIG.61.

3. Construction of Human CDR-Grafted Antibody H Chain and L Chain TandemExpression Vector

A tandem expression vector containing both of cDNA coding for humanCDR-grafted antibody H chain and cDNA coding for human CDR-graftedantibody L chain was constructed in the following manner (FIG. 62 andFIG. 63).

Three μg of phKM796HMS1, obtained in Paragraph 2(1) of Example 2, weredissolved in 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 mMDTT, 1 unit of SalI was added thereto and the mixture was allowed toreact at 37° C. for 10 minutes to effect partial digestion. Theresulting mixture was subjected to ethanol precipitation and thethus-obtained precipitate was dissolved in 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Ten units of MluI was added theretoto allow the mixture to react at 37° C. for 1 hour. The reaction mixturewas fractionated by agarose gel electrophoresis to recover about 0.2 μgof a DNA fragment of about 5.9 kb. Then, about 2 μg of pAGE107 asdescribed in EP-A-0 405 285 was added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, 10 units of MluI and 10 units ofSalI were added thereto, and the mixture was allowed to react at 37° C.for 1 hour. The reaction mixture was fractionated by agarose gelelectrophoresis to recover about 0.2 μg of a DNA fragment of about 3.35kb. Then, 0.1 μg of the thus-obtained MluI-SalI fragment of phKM796HMS1was ligated to 0.1 μg of the MluI-SalI fragment of pAGE107 using a DNAligation kit (Takara Shuzo). The thus-obtained recombinant plasmid DNAwas used to transform Escherichia coli HB101 to obtain the plasmidphKM796H107 shown in FIG. 62.

Then, 3 μg of phKM796H107 were added to 30 μl of 10 mMTris-hydrochloride (pH 7.5) containing 10 mM magnesium chloride, 100 mMsodium chloride and 1 mM DTT, 10 units of ClaI was added thereto and themixture was allowed to react at 37° C. for 1 hour. The reaction mixturewas subjected to phenol-chloroform extraction and ethanol precipitation.The resulting precipitate was dissolved in 20 μl of DNA polymerase Ibuffer, 5 units of Escherichia coli-derived DNA polymerase I Klenowfragment were added, and the 5' cohesive ends produced by ClaI digestionwere rendered blunt by incubation at 22° C. for 30 minutes. The reactionmixture was fractionated by agarose gel electrophoresis to recover about0.2 μg of a DNA fragment of about 3.35 kb. The reaction mixture was alsosubjected to phenol-chloroform extraction and then to ethanolprecipitation. The resulting precipitate was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, 10 units of MluI were added theretoand the mixture was allowed to react at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis to recover about0.3 μg of a DNA fragment of about 7.5 kb. Separately, 3 μg ofphKM796LMS1 were added to 30 μl of 50 mM Tris-hydrochloride buffer (pH7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and 1mM DTT, 10 units of XhoI were added and the mixture was allowed to reactat 37° C. for 1 hour. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation. Theresulting precipitate was dissolved in 20 μl of DNA polymerase I buffer,5 units of Escherichia coli-derived DNA polymerase I Klenow fragmentwere added, and the 5' cohesive ends produced by XhoI digestion wererendered blunt by incubation at 22° C. for 30 minutes. The reactionmixture was subjected to phenol-chloroform extraction followed byethanol precipitation. The resulting precipitate was added to 30 μl of10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT, 10 units of MluI wereadded thereto, and the mixture was allowed to react at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis to recover about 0.3 μg of a DNA fragment of about 9.3kb. Then, 0.1 μg of the thus-obtained MluI-ClaI fragment of phKM796H107was ligated to 0.1 μg of the MluI-XhoI fragment of phKM796LMS1 using aDNA ligation kit (Takara Shuzo). The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 to obtain the plasmidphKM796HL1 shown in FIG. 63.

4. Expression of Human CDR-Grafted Anti-GM₂ Antibody in YB2/0 Cells

The plasmids were introduced into YB2/0 cells by the electroporationmethod of Miyaji et al. [Cytotechnology, 3, 133 (1990)].

After introduction of 4 μg of phKM796HL1 obtained in Paragraph 3 ofExample 2 into 4×10⁶ YB2/0 (ATCC CRL1581) cells, the cells weresuspended in 40 ml of RPMI1640-FCS(10) [RPMI1640 medium (NissuiPharmaceutical) containing 10% of FCS, 1/4 volume of 7.5% NaHCO₃, 3% of200 mM L-glutamine solution (Gibco) and 0.5% of penicillin-streptomycinsolution (Gibco; containing 5,000 units/ml penicillin and 5,000 μg/mlstreptomycin)], and the suspension was distributed in 200-μl portionsinto wells of 96-well microtiter plates. After 24 hours of incubation at37° C. in a CO₂ incubator, G418 (Gibco) was added to a concentration of0.5 mg/ml and then incubation was continued for 1 to 2 weeks.Transformant colonies appeared, the culture fluid was recovered fromeach well in which the cells had grown to confluence and anenzyme-linked immunosorbent assay (ELISA) described in Paragraph 11 ofExample 1 was conducted for anti-GM₂ human CDR-grafted antibody activitymeasurement.

The clone showing the highest activity in ELISA among the clonesobtained gave a human CDR-grafted anti-GM₂ antibody content of about 0.1μg/ml of culture fluid.

Cells of the clone showing the above-mentioned human CDR-graftedanti-GM₂ antibody activity were suspended in RPMI1640-FCS(10) mediumcontaining 0.5 mg/ml G418 and 50 nM MTX to a concentration of 1 to 2×10⁵cells/ml, and the suspension was distributed in 2-ml portions into wellsof 24-well plates. Incubation was performed at 37° C. in a CO₂ incubatorfor 1 to 2 weeks to induce 50 nM MTX-resistant clones. At the time ofconfluence, the human CDR-grafted anti-GM₂ antibody activity in eachculture fluid was determined by ELISA. The 50 nM MTX-resistant cloneshowing the highest activity among the clones obtained showed a humanCDR-grafted anti-GM₂ antibody content of about 1.0 μg/ml.

Cells of the above 50 nM MTX-resistant clone were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml, and the suspension was distributedin 2-ml portions into wells of 24-well plates. Incubation was carriedout at 37° C. in a CO₂ incubator for 1 to 2 weeks to induce 200 nMMTX-resistant clones. At the time of confluence, each culture fluid wasassayed for human CDR-grafted anti-GM₂ antibody activity by ELISA. The200 nM MTX-resistant clone showing the highest activity among the clonesobtained had a human CDR-grafted anti-GM₂ antibody content of about 5.0μg/ml.

As described in detail hereinabove, the present invention provideshumanized antibodies reacting with the ganglioside GM₂.

EXAMPLE 3 Production of Human CDR-Grafted Anti-GM₂ Antibodies (2)

1. Construction of Tandem Cassette Type Humanized Antibody ExpressionVector, pKANTEX93

A tandem cassette type humanized antibody expression vector, pKANTEX93,for the expression of a human CDR-grafted antibody in mammalian cellswas constructed based on the plasmid pSE1UK1SEd1-3 described inJP-A-2-257891 by inserting a DNA fragment coding for a human CDR-graftedanti-GM₂ antibody H chain variable region and a DNA fragment coding fora human CDR-grafted anti-GM₂ antibody L chain variable region into saidplasmid upstream of the human antibody γ 1H chain constant region cDNAand human antibody _(K) L chain constant region cDNA, respectively, inthe following manner.

(1) Modification of ApaI and EcoRI Restriction Enzyme Sites Occurring inRabbit β-Globin Gene Splicing and Poly A Signals

For making it possible to construct a human CDR-grafted antibodyexpression vector by inserting human CDR-grafted antibody variableregions cassette-wise in the form of NotI-ApaI (H chain) and EcoRI-SplI(L chain) restriction fragments into a vector for human CDR-graftedantibody expression, the ApaI and EcoRI restriction sites occurring inthe rabbit β-globin gene splicing and poly A signals of the plasmidpSE1UK1SEd1-3 were modified in the following manner.

Three μg of the plasmid pBluescript SK(-) (Stratagene) was added to 10μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride and 1 mM DTT, 10 units of the restriction enzyme ApaI(Takara Shuzo) was further added, and the digestion reaction was allowedto proceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, and the 3' cohesive ends resulting from ApaIdigestion were rendered blunt using DNA Blunting Kit (Takara Shuzo),followed by ligation using DNA Ligation Kit (Takara Shuzo). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101. Thus was obtained a plasmid, pBSA, shown in FIG.64. Furthermore, 3 μg of the plasmid PBSA thus obtained was added to 10μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units of therestriction enzyme EcoRI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, and the 5' cohesive endsresulting from EcoRI digestion were rendered blunt using DNA BluntingKit (Takara Shuzo), followed by ligation using DNA Ligation Kit (TakaraShuzo). The thus-obtained recombinant plasmid DNA solution was used totransform Escherichia coli HB101. Thus was obtained the plasmid PBSAEshown in FIG. 65.

Then, 3 μg of the thus-obtained plasmid pBSAE was added to 10 μl of 10mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT, 10 units of therestriction enzyme HindIII (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 20 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, and the solution wasdivided into two 10-μl portions. To one portion, 10 units of therestriction enzyme SacII (Toyobo) was further added and, to the other,10 units of the restriction enzyme KpnI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour.Both the reaction mixtures were fractionated by agarose gelelectrophoresis, whereby about 0.3 μg each of a HindIII-SacII fragment(about 2.96 kb) and a KpnI-HindIII fragment (about 2.96 kb) wererecovered.

Then, 3 μg of the plasmid pSE1UK1SEd1-3 was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of the restriction enzyme SacII (Toyobo) and 10units of the restriction enzyme KpnI (Takara Shuzo) were further added,and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was subjected to ethanol precipitation, the precipitatewas dissolved in 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT,10 units of the restriction enzyme HindIII (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis,whereby about 0.2 μg each of a HindIII-SacII fragment (about 2.42 kb)and a KpnI-HindIII fragment (about 1.98 kb) were recovered.

Then, 0.1 μg of the thus-obtained HindIII-SacII fragment ofpSE1UK1SEd1-3 and 0.1 μg of the above HindIII-SacII fragment of pBSAEwere dissolved in a total of 20 μl of sterilized water and ligated toeach other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101 and, as a result, a plasmid, pBSH-S, shown inFIG. 66 was obtained. Furthermore, 0.1 μg of the above-mentionedXpnI-HindIII fragment of pSE1UK1SEd1-3 and 0.1 μg of the above-mentionedKpnI-HindIII fragment of pBSAE were dissolved in a total of 20 μl ofsterilized water and ligated to each other using Ready-To-Go T4 DNALigase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNAsolution was used to transform Escherichia coli HB101, and the plasmidpBSK-H shown in FIG. 67 was obtained.

Then, 3 μg each of the thus-obtained plasmids pBSH-S and pBSK-H wererespectively added to 10-μl portions of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units ofthe restriction enzyme ApaI (Takara Shuzo) was further added to eachmixture, and the reaction was allowed to proceed at 37° C. for 1 hour.Both the reaction mixtures were subjected to ethanol precipitation. Witheach precipitate, the 3' cohesive ends resulting from ApaI digestionwere rendered blunt using DNA Blunting Kit (Takara Shuzo), followed byligation using DNA Ligation Kit (Takara Shuzo). The thus-obtainedrecombinant DNA solution were used to transform Escherichia coli HB101,and the plasmids PBSH-SA and pBSK-HA shown in FIG. 68 were obtained.

Then, 5 μg each of the thus-obtained plasmids pBSH-SA and pBSK-HA wererespectively added to 10-μl portions of 50 mM Tris-hydrochloride buffer(pH 7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and1 mM DTT, 1 unit of the restriction enzyme EcoRI (Takara Shuzo) wasfurther added to each mixture, and the reaction was allowed to proceedat 37° C. for 10 minutes for partial digestion. Both the reactionmixtures were subjected to ethanol precipitation. With each precipitate,the 5' cohesive ends resulting from EcoRI digestion were rendered bluntusing DNA Blunting Kit (Takara Shuzo), followed by fractionation byagarose gel electrophoresis, whereby about 0.5 μg each of a fragmentabout 5.38 kb in length and a fragment about 4.94 kb in length wererecovered. The thus-recovered fragments (0.1 μg each) were eachdissolved in a total of 20 μl of sterilized water and subjected toligation treatment using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech).The thus-obtained recombinant DNA solutions were respectively used totransform Escherichia coli HB101, and the plasmids PBSH-SAE and pBSK-HAEshown in FIG. 69 were obtained.

Then, 3 μg each of the thus-obtained plasmids PBSH-SAE and pBSK-HAE wererespectively added to 10-μl portions of 50 mM Tris-hydrochloride buffer(pH 7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and1 mM DTT, 10 units of the restriction enzyme EcoRI (Takara Shuzo) wasfurther added to each mixture, and the reaction was allowed to proceedat 37° C. for 1 hour. Both the reaction mixtures were subjected toethanol precipitation. With each precipitate, the 5' cohesive endsresulting from EcoRI digestion were rendered blunt using DNA BluntingKit (Takara Shuzo), followed by ligation using DNA Ligation Kit (TakaraShuzo). The thus-obtained recombinant plasmid DNA solutions were eachused to transform Escherichia coli HB101, and two plasmids, PBSH-SAEEand pBSK-HAEE, shown in FIG. 70 were obtained. Ten μg each of thethus-obtained plasmids were subjected to sequencing reaction accordingto the instructions attached to AutoRead Sequencing Kit (PharmaciaBiotech), followed by base sequence determination by electrophoresis onA.L.F. DNA Sequencer (Pharmacia Biotech), whereby it was confirmed thatboth the ApaI and EcoRI sites had disappeared as a result of the abovemodification.

(2) SalI Restriction Site Introduction Downstream From Rabbit β-GlobinGene Splicing and Poly A Signals and SV40 Early Gene Poly A Signal

For making it possible to exchange the antibody H chain and L chainexpression promoters of the human CDR-grafted antibody expression vectoreach for an arbitrary promoter, a SalI restriction site was introducedinto the plasmid pSE1UK1SEd1-3 downstream from the rabbit β-globin genesplicing and poly A signals and from the SV40 early gene poly A signalin the following manner.

Three μg of the plasmid pBSK-HAEE obtained in Paragraph 1 (1) of Example3 was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme NaeI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 20 μl of 50 mM Tris-hydrochloride buffer (pH 9.0)containing 1 mM magnesium chloride, 1 unit of alkaline phosphatase (E.coli C75, Takara Shuzo) was further added, and the reaction was allowedto proceed at 37° C. for 1 hour for dephosphorylation at the 5' termini.The reaction mixture was further subjected to phenol-chloroformextraction and then to ethanol precipitation, and the precipitate wasdissolved in 20 μl of 10 mM Tris-hydrochloride buffer (pH 8.0)containing 1 mM disodium ethylenediaminetetraacetate (hereinafterbriefly referred to as TE buffer). One μl of said reaction solution and0.1 μg of a phosphorylated SalI linker (Takara Shuzo) were added tosterilized water to make a total volume of 20 μl, followed by ligationtreatment using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and a plasmid, pBSK-HAEESal, shown in FIG. 71was obtained. Ten μg of the plasmid thus obtained was subjected tosequencing reaction according to the instructions attached to AutoReadSequencing Kit (Pharmacia Biotech), followed by electrophoresis onA.L.F. DNA Sequencer (Pharmacia Biotech) for base sequencedetermination, whereby it was confirmed that one SalI restriction sitehad been introduced downstream from the rabbit β-globin gene splicingand poly A signals and from the SV40 early gene poly A signal.

(3) Modification of ApaI Restriction Site Occurring in Poly A Signal ofHerpes Simplex Virus Thymidine Kinase (Hereinafter Referred to as HSVtk)Gene

The ApaI restriction site occurring in the HSVtk gene poly A signaldownstream from the Tn5 kanamycin phosphotransferase gene of the plasmidpSE1UK1SEd1-3 was modified in the following manner.

Three μg of the plasmid PBSA obtained in Paragraph 1 (1) of Example 3was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme SacII (Toyobo) was further added, and the reactionwas allowed to proceed at 37° C. for 1 hour. The reaction mixture wassubjected to ethanol precipitation, the precipitate was added to 10 μlof 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodiumchloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme XhoI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, whereby about 1μg of a SacII-XhoI fragment (about 2.96 kb) was recovered.

Then, 5 μg of the plasmid pSE1UK1SEd1-3 was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of the restriction enzyme SacII (Toyobo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was subjected to ethanol precipitation, theprecipitate was added to 10 μl of 50 mM Tris-hydrochloride buffer (pH7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride and 1mM DTT, 10 units of the restriction enzyme XhoI (Takara Shuzo) wasfurther added, and the reaction was fractionated by agarose gelelectrophoresis, whereby about 1 μg of a SacII-XhoI fragment (about 4.25kb) was recovered.

Then, 0.1 μg of the above SacII-XhoI fragment of PBSA and the aboveSacII-XhoI fragment of pSE1UK1SEd1-3 were added to a total of 20 μl ofsterilized water, followed by ligation using Ready-To-Go T4 DNA Ligase(Pharmacia Biotech). The thus-obtained recombinant plasmid DNA solutionwas used to transform Escherichia coli HB101, and the plasmid pBSX-Sshown in FIG. 72 was obtained.

Then, 3 μg of the thus-obtained plasmid pBSX-S was added to 10 μl of 10mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride and 1 mM DTT, 10 units of the restriction enzyme ApaI (TakaraShuzo) was further added, and the reaction was allowed to proceed at 37°C. for 1 hour. The reaction mixture was subjected to ethanolprecipitation, the 3' cohesive ends resulting from ApaI digestion wererendered blunt using DNA Blunting Kit (Takara Shuzo) and then ligationwas carried out using DNA Ligation Kit (Takara Shuzo). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and a plasmid, pBSX-SA, shown in FIG. 73 was obtained. Ten μg ofthe thus-obtained plasmid was subjected to sequencing reaction accordingto the instructions attached to AutoRead Sequencing Kit (PharmaciaBiotech), followed by electrophoresis on A.L.F. DNA Sequencer (PharmaciaBiotech) for base sequence determination, whereby it was confirmed thatthe ApaI restriction site in the HSVtk gene poly A signal haddisappeared.

(4) Construction of Human CDR-Grafted Antibody L Chain Expression Unit

A plasmid, pMohC_(K), containing a human antibody _(K) L chain constantregion cDNA downstream from the promoter/enhancer of the Moloney mouseleukemia virus long terminal repeat and having a human CDR-graftedantibody L chain expression unit allowing cassette-wise insertion thereinto of a human CDR-grafted antibody L chain variable region wasconstructed in the following manner.

Three μg of the plasmid pBluescript SK(-) (Stratagene) was added to 10μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride and 1 mM DTT, 10 units of the restriction enzyme SacI(Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, the precipitate was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 50 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzymeClaI (Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, and the cohesive ends resulting from SacI andClaI digestion were rendered blunt using DNA Blunting Kit (TakaraShuzo), followed by fractionation by agarose gel electrophorsis, wherebyabout 1 μg of a DNA fragment about 2.96 kb in length was recovered. A0.1-μg portion of the DNA fragment recovered was added to a total of 20μl of sterilized water and subjected to ligation reaction usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pBSSC shown in FIG. 74 was obtained.

Then, 3 μg of the thus-obtained plasmid pBSSC was added to 10 μl of 10mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride and 1 mM DTT, 10 units of the restriction enzyme KpnI (TakaraShuzo) was further added, and the reaction was allowed to proceed at 37°C. for 1 hour. The reaction mixture was subjected to ethanolprecipitation, the precipitate was dissolved in 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzymeXhoI (Takara Shuzo) was further added, and the reaction was allowed toproceed at 37+ C. for 1 hour. The reaction mixture was fractionated byagarose gel electrophoresis, whereby about 1 μg of a KpnI-XhoI fragment(about 2.96 kb) was recovered.

Then, 5 μg of the plasmid pAGE147 obtained in Paragraph 7 (1) of Example1 was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme KpnI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme XhoI (Takara Shuzo) was furtheradded, and the reaction was fractionated by agarose gel electrophoresis,whereby about 0.3 μg of a KpnI-XhoI fragment (about 0.66 kb) containingthe Moloney mouse leukemia virus long terminal repeat promoter/enhancerwas recovered.

Then, 0.1 μg of the KpnI-XhoI fragment of pBSSC and 0.1 μg of theKpnI-XhoI fragment of pAGE147 each obtained as mentioned above weredissolved in a total of 20 μl of sterilized water and subjected toligation using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pBSMo shown in FIG. 75 wasobtained.

Then, 3 μg of the above plasmid pBSMo was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of the restriction enzyme KpnI (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was subjected to ethanol precipitation, theprecipitate was dissolved in 10 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 50 mM sodium chloride, 10 mM magnesium chloride and1 mM DTT, 10 units of the restriction enzyme HindIII (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, whereby about 1 μg of a KpnI-HindIII fragment (about3.62 kb) was recovered.

Then, synthetic DNAs respectively having the base sequences shown in SEQID No:38 and SEQ ID No:39 were synthesized using an automatic DNAsynthesizer (Applied Biosystems model 380A). To 15 μl of sterilizedwater were added 0.3 μg each of the thus-obtained synthetic DNAs, andthe mixture was heated at 65° C. for 5 minutes. The reaction mixture wasallowed to stand at room temperature for 30 minutes and then 2 μl of10-fold concentrated buffer [500 mM Tris-hydrochloride (pH 7.6), 100 mMmagnesium chloride, 50 mM DTT] and 2 μl of 10 mM ATP were added, 10units of T4 polynucleotide kinase was further added, and the reactionwas allowed to proceed at 37° C. for 30 minutes for phosphorylation ofthe 5' termini. To a total of 20 μl of sterilized water were added 0.1μg of the above KpnI-HindIII fragment (3.66 kb) derived from the plasmidpBSMo and 0.05 μg of the phsophorylated synthetic DNA pair, and ligationwas effected using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pBSMoS shown in FIG. 76 wasobtained. Ten μg of the plasmid thus obtained was subjected tosequencing reaction according to the instructions attached to AutoReadSequencing Kit (Pharmacia Biotech), followed by electrophoresis onA.L.F. DNA Sequencer (Pharmacia Biotech) for base sequencedetermination, whereby it was confirmed that the synthetic DNA pair hadbeen introduced as desired.

Then, 3 μg of the plasmid pChiIgLA1 described in JP-A-5-304989 wasdissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units each of the restriction enzymes EcoRI (Takara Shuzo) andEcORV (Takara Shuzo) were further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was fractionated byagarose gel electrophoresis, whereby about 1 μg of an EcoRI-EcoRVfragment (about 9.70 kb) was recovered.

Then, synthetic DNAs respectively having the base sequences shown in SEQID NO:40 and SEQ ID NO:41 were synthesized using an automatic DNAsynthesizer (Applied Biosystems model 380A). To 15 μl of sterilizedwater were added 0.3 μg each of the thus-obtained synthetic DNAs, andthe mixture was heated at 65° C. for 5 minutes. The reaction mixture wasallowed to stand at room temperature for 30 minutes. Then, 2 μl of10-fold concentrated buffer [500 mM Tris-hydrochloride (pH 7.6), 100 mMmagnesium chloride, 50 mM DTT] and 2 μl of 10 mM ATP were added, 10units of T4 polynucleotide kinase was further added, and the reactionwas allowed to proceed at 37° C. for 30 minutes for phosphorylation ofthe 5' termini. To a total of 20 μl of sterilized water were added 0.1μg of the above EcoRI-EcORV fragment (9.70 kb) derived from the plasmidpChiIgLA1 and 0.05 μg of the phsophorylated synthetic DNA, and ligationwas effected using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pChiIgLA1S shown in FIG. 77 wasobtained.

Then, 3 μg of the plasmid pBSMoS obtained in the above manner wasdissolved in 10 μl of 20 mM Tris-hydrochloride buffer (pH 8.5)containing 100 mM potassium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme HpaI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was subjected to ethanol precipitation, the precipitatewas dissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme EcoRI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis,whereby about 1 μg of an HpaI-EcoRI fragment (about 3.66 kb) wasrecovered.

Then, 10 μg of the plasmid pChiIgLA1S obtained as mentioned above wasdissolved in 10 μl of 20 mM Tris-acetate buffer (pH 7.9) containing 50mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT and 100 μg/mlBSA, 10 units of the restriction enzyme NlaIV (New England BioLabs) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was subjected to ethanol precipitation, theprecipitate was dissolved in 10 μl of 50 mM Tris-hydrochloride buffer(pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride and1 mM DTT, 10 units of the restriction enzyme EcoRI (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, whereby about 0.3 μg of an NlaIV-EcoRI fragment (about0.41 kb) was recovered.

Then, 0.1 μg of the above HpaI-EcoRI fragment of pBSMoS and 0.1 μg ofthe above NlaIV-EcoRI fragment of pChiIgLA1S were added to a total of 20μl of sterilized water, and ligation was effected using Ready-To-Go T4DNA Ligase (Pharmacia Biotech). The thus-obtained recombinant plasmidDNA solution was used to transform Escherichia coli HB101, and theplasmid pMohCK shown in FIG. 78 was obtained.

(5) Construction of Human CDR-Grafted Antibody H Chain Expression Unit

A plasmid, pMohCγ1, containing a human antibody γ1 H chain constantregion cDNA downstream from the promoter/enhancer of the Moloney mouseleukemia virus long terminal repeat and having a human CDR-graftedantibody H chain expression unit allowing cassette-wise insertion thereinto of a human CDR-grafted antibody H chain variable region wasconstructed in the following manner.

Three μg of the plasmid pBSMo obtained in Paragraph 1 (4) of Example 3was added to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme XhoI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was subjected to ethanol precipitation, the precipitatewas dissolved in 10 μl of 30 mM sodium acetate buffer (pH 5.0)containing 100 mM sodium chloride, 1 mM zinc acetate and 10% glycerol,10 units of Mung bean nuclease (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 10 minutes. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation, the cohesive ends of the precipitate wererendered blunt using DNA Blunting Kit (Takara Shuzo) and ligation waseffected using DNA Ligation Kit (Takara Shuzo). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pBSMoSal shown in FIG. 79 was obtained. A 10-μgportion of the plasmid obtained was subjected to sequencing reactionaccording to the instructions attached to AutoRead Sequencing Kit(Pharmacia Biotech), followed by electrophoresis on A.L.F. DNA Sequencer(Pharmacia Biotech) for base sequence determination, whereby it wasconfirmed that the XhoI restriction site upstream of the Moloney mouseleukemia virus long terminal repeat promoter/enhancer had disappeared.

Then, 3 μg of the plasmid pBSMoSal obtained as mentioned above was addedto 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride and 1 mM DTT, 10 units of the restriction enzyme KinI(Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, the precipitate was dissolved in 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 50 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzymeHindIII (Takara Shuzo) was further added, and the reaction was allowedto proceed at 37° C. for 1 hour. The reaction mixture was fractionatedby agarose gel electrophoresis, whereby about 1 μg of a KpnI-HindIIIfragment (about 3.66 kb) was recovered.

Then, synthetic DNAs respectively having the base sequences shown in SEQID NO:42 and SEQ ID NO:43 were synthesized using an automatic DNAsynthesizer (Applied Biosystems model 380A). To 15 μl of sterilizedwater were added 0.3 μg each of the thus-obtained synthetic DNAs, andthe mixture was heated at 65° C. for 5 minutes. The reaction mixture wasallowed to stand at room temperature for 30 minutes. Then, 2 μl of10-fold concentrated buffer [500 mM Tris-hydrochloride (pH 7.6), 100 mMmagnesium chloride, 50 mM DTT] and 2 μl of 10 mM ATP were added, 10units of T4 polynucleotide kinase was further added, and the reactionwas allowed to proceed at 37° C. for 30 minutes for phosphorylation ofthe 5' termini. To a total of 20 μl of sterilized water were added 0.1μg of the above KpnI-HindIII fragment (3.66 kb) derived from the plasmidpBSMoSal and 0.05 μg of the phosphorylated synthetic DNA, and ligationwas effected using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pBSMoSalS shown in FIG. 80 wasobtained. A 10-μg portion of the thus-obtained plasmid was subjected tosequencing reaction according to the instructions attached to AutoReadSequencing Kit (Pharmacia Biotech), followed by electrophoresis onA.L.F. DNA Sequencer (Pharmacia Biotech), for base sequencedetermination whereby it was confirmed that the synthetic DNA had beenintroduced as desired.

Then, 10 μg of the plasmid pChiIgHB2 described in JP-A-5-304989 wasdissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme Eco52I (Toyobo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was subjected to ethanol precipitation, the precipitatewas dissolved in 10 μl of 30 mM sodium acetate buffer (pH 5.0)containing 100 mM sodium chloride, 1 mM zinc acetate and 10% glycerol,10 units of Mung bean nuclease (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 10 minutes. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation, and the cohesive ends were rendered blunt usingDNA Blunting Kit (Takara Shuzo). After ethanol precipitation, theprecipitate was dissolved in 10 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units ofthe restriction enzyme ApaI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, whereby about0.7 μg of ApaI-blunt end fragment (about 0.99 kb) was recovered.

Then, 3 μg of the plasmid pBluescript SK(-) (Stratagene) was added to 10μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride and 1 mM DTT, 10 units of the restriction enzyme ApaI(Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, the precipitate was dissolved in 10 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 100 μg/ml BSA, 10 units of therestriction enzyme SmaI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, whereby about 1μg of an ApaI-SmaI fragment (about 3.0 kb) was recovered.

Then, 0.1 μg of the ApaI-blunt end fragment of pChiIgHB2 and 0.1 μg ofthe ApaI-SmaI fragment of pBluescript SK(-), each obtained as mentionedabove, were added to a total of 20 μl of sterilized water and ligated toeach other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pBShCγ1 shown in FIG. 81 wasobtained.

Then, 5 μg of the above plasmid pBShCγ1 was dissolved in 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of the restriction enzyme ApaI (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was subjected to ethanol precipitation, theprecipitate was dissolved in 10 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 50 mM sodium chloride, 10 mM magnesium chloride and1 mM DTT, 10 units of the restriction enzyme SpeI (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, whereby about 1 μg of an ApaI-SpeI fragment (about 1.0kb) was recovered.

Then, 3 μg of the plasmid pBSMoSa1S obtained as mentioned above wasdissolved in 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme ApaI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 50 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT,10 units of the restriction enzyme SpeI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis,whereby about 1 μg of an ApaI-SpeI fragment (about 3.66 kb) wasrecovered.

Then, 0.1 μg of the ApaI-SpeI fragment of pBShCγ1 and 0.1 μg of theApaI-SpeI fragment of pBSMoSa1S, each obtained as mentioned above, wereadded to a total of 20 μl of sterilized water and ligated to each otherusing Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pMohCγ1 shown in FIG. 82 was obtained.

(6) Construction of Tandem Cassette Type Humanized Antibody ExpressionVector pKANTEX93

A tandem cassette type humanized antibody expression vector, pKANTEX93,was constructed using the various plasmids obtained in Paragraphs (1)through (5) of Example 3 in the following manner.

Three μg of the plasmid pBSH-SAEE obtained in Paragraph 1 (1) of Example3 was added to 10 μl 10 mM Tris-hydrochloride buffer (pH 7.5) containing50 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 unitsof the restriction enzyme HindIII (Takara Shuzo) was further added, andthe reaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme SalI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis,whereby about 1 μg of a HindIII-SalI fragment (about 5.42 kb) wasrecovered.

Then, 5 μg of the plasmid pBSK-HAEE obtained in Paragraph 1 (1) ofExample 3 was added to 10 μl of 10 mM Tris-hydrochloride buffer (ph 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme KpnI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 50 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT,10 units of the restriction enzyme HindIII (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis,whereby about 0.8 μg of a KpnI-HindIII fragment (about 1.98 kb)containing the rabbit β-globin gene splicing and poly A signals, theSV40 early gene poly A signal and the SV40 early gene promoter wasrecovered.

Then, 5 μg of the plasmid pMohcyl obtained in Paragraph 1 (5) of Example3 was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme KpnI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme SalI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis,whereby about 0.8 μg of a human CDR-grafted antibody H chain expressionunit-containing KpnI-SalI fragment (about 1.66 kb) was recovered.

Then, 0.1 μg of the HindIII-SalI fragment of pBSH-SAEE, 0.1 μg of theKpnI-HindIII fragment of pBSK-HAEE and 0.1 μg of the KpnI-SalI fragmentof pMohCγ1, each obtained as mentioned above, were added to a total of20 μl of sterilized water and ligated together using Ready-To-Go T4 DNALigase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNAsolution was used to transform Escherichia coli HE101, and the plasmidpMoγ1SP shown in FIG. 83 was obtained.

The, 3 μg of the above plasmid pMoγ1SP was added to 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzymeSalI (Takara Shuzo) and 10 units of the restriction enzyme XhoI werefurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, whereby about 1 μg of a SalI-XhoI fragment (about 9.06kb) was recovered.

Then, 5 μg of the plasmid pBSK-HAEESal obtained in Paragraph 1 (2) ofExample 3 was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme KpnI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme SalI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis,whereby about 0.7 μg of a KpnI-SalI fragment (about 1.37 kb) containingthe rabbit β-globin gene splicing and poly A signals and the SV40 earlygene poly A signal was recovered.

Then, 5 μg of the plasmid pMohC_(K) obtained in Paragraph 1 (4) ofExample 3 was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1mM DTT, 10 units of therestriction enzyme KpnI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasdissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme XhoI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis,whereby about 0.7 μg of a human CDR-grafted antibody L chain expressionunit-containing KpnI-XhoI fragment (about 1.06 kb) was recovered.

Then, 0.1 μg of the SalI-XhoI fragment of pMoγ1SP, 0.1 μg of theKpnI-SalI fragment of pBSK-HAEESal and 0.1 μg of the KpnI-XhoI fragmentof pMohC_(K), each obtained as mentioned above, were added to a total of20 μl of sterilized water and ligated together using Ready-To-Go T4 DNALigase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNAsolution was used to transform Escherichia coli HB101, and the plasmidPMO_(K) γ1SP shown in FIG. 84 was obtained.

Then, 3 μg of the above plasmid pMO_(K) γ1SP was added to 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzymeXhoI (Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, the precipitate was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 1 units of the restriction enzyme SacII (Toyobo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 10minutes for partial digestion. The reaction mixture was fractionated byagarose gel electrophoresis, and about 0.2 μg of a SacII-XhoI fragment(about 8.49 kb) was recovered.

Then, 3 μg of the plasmid pBSX-SA obtained in Paragraph 1 (4) of Example3 was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme SacII (Toyobo) was further added, and the reactionwas allowed to proceed at 37° C. for 1 hour. The reaction mixture wassubjected to ethanol precipitation, the precipitate was dissolved in 10μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodiumchloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme XhoI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, and about 1 μgof a SacII-XhoI fragment (about 4.25 kb) was recovered.

Then, 0.1 μg of the SacII-XhoI fragment of PMO_(K) γ1SP and 0.1 μg ofthe SacII-XhoI fragment of pBSX-SA, each obtained as mentioned above,were added to a total of 20 μl of sterilized water and ligated to eachother using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pKANTEX93 shown in FIG. 85 wasobtained.

2. Expression of Human Anti-GM₂ Chimera Antibody Using HumanizedAntibody Expression Vector pKANTEX93

Human anti-GM₂ chimera antibody expression was effected using thehumanized antibody expression vector pKANTEX93 mentioned above inParagraph 1 of Example 3 in the following manner.

(1) Construction of Plasmid pBSH3 Containing Mouse Anti-GM₂ AntibodyKM796 H Chain Variable Region cDNA

Three μg of the plasmid pBluescript SK(-) (Stratagene) was added to 10μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride and 1 mM DTT, 10 units each of the restrictionenzymes SacII (Toyobo) and KpnI (Takara Shuzo) were further added, andthe reaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, and the precipitate wassubjected to blunting treatment for rendering blunt the 3' cohesive endsresulting from the restriction enzyme digestion using DNA Blunting Kit(Takara Shuzo) and then to fractionation by agarose gel electrophoresis,and about 1 μg of a DNA fragment about 2.95 kb in size was recovered.

Then, synthetic DNAs respectively having the base sequences shown in SEQID NO:44 and SEQ ID NO:45 were synthesized using an automatic DNAsynthesizer (Applied Biosystems model 380A). To 15 μl of sterilizedwater were added 0.3 μg each of the synthetic DNAs obtained, and themixture was heated at 65° C. for 5 minutes. The reaction mixture wasallowed to stand at room temperature for 30 minutes and then 2 μl of10-fold concentrated buffer [500 mM Tris-hydrochloride (pH 7.6), 100 mMmagnesium chloride, 50 mM DTT] and 2 μl of 10 mM ATP were added, 10units of T4 polynucleotide kinase was further added, and the reactionwas allowed to proceed at 37° C. for 30 minutes for phosphorylating the5' termini. To a total of 20 μl of sterilized water were added 0.1 μg ofthe DNA fragment (2.95 kb) derived from the plasmid pBluescript SK(-)and 0.05 μg of the phosphorylated synthetic DNA, each obtained asmentioned above, followed by ligation to each other using Ready-To-Go T4DNA Ligase (Pharmacia Biotech). The thus-obtained recombinant plasmidDNA solution was used to transform Escherichia coli HN101, and theplasmid pBSNA shown in FIG. 86 was obtained. Ten μg of the plasmidobtained was subjected to sequencing reaction treatment according to theinstructions attached to AutoRead Sequencing Kit (Pharmacia Biotech),followed by electrophoresis on A.L.F. DNA Sequencer (Pharmacia Biotech)for base sequence determination, whereby it was confirmed that thesynthetic DNA had been introduced as desired.

Then, 3 μg of the plasmid pBSNA obtained as mentioned above was added to10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride and 1 mM DTT, 10 units of the restriction enzyme ApaI(Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, the precipitate was added to 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride, 1 mM DTT, 100 μg/ml BSA and 0.01% Triton X-100,10 units of the restriction enzyme NotI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis, andabout 1 μg of a DNA fragment about 2.95 kb in size was recovered.

Then, 10 μg of the plasmid pChi796HM1 obtained in Paragraph 7 (3) ofExample 1 was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme ApaI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasadded to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100 μg/mlBSA and 0.01% Triton X-100, 10 units of the restriction enzyme NotI(Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was fractionated byagarose gel electrophoresis, and about 0.3 μg of a DNA fragment about0.45 kb in size was recovered.

Then, 0.1 μg of the Apa-NotI fragment of pBSNA and 0.1 μg of theApa-NotI fragment of pChi796HM1, each obtained as mentioned above, wereadded to a total of 20 μl of sterilized water and ligated to each otherusing Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pBSH3 shown in FIG. 87 was obtained.

(2) Construction of Plasmid pBSL3 Containing Mouse Anti-GM₂ AntibodyKM796 L Chain Variable Region cDNA

Three μg of the plasmid pbluescript SK(-) (Stratagene) was added to 10μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride and 1 mM DTT, 10 units of the restriction enzyme KpnI(Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, and the precipitate was subjected to bluntingtreatment for rendering blunt the 3' cohesive ends resulting from KpnIdigestion using DNA Blunting Kit (Takara Shuzo) and then to ethanolprecipitation, the precipitate was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of the restriction enzyme SacI (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, whereby about 1 μg of a DNA fragment about 2.95 kb insize was recovered.

Then, synthetic DNAs respectively having the base sequences shown in SEQID NO:46 and SEQ ID NO:47 were synthesized using an automatic DNAsynthesizer (Applied Biosystems model 380A). To 15 μl of sterilizedwater were added 0.3 μg each of the synthetic DNAs obtained, and themixture was heated at 65° C. for 5 minutes. The reaction mixture wasallowed to stand at room temperature for 30 minutes. Then, 2 μl of10-fold concentrated buffer [500 mM Tris-hydrochloride (pH 7.5), 100 mMmagnesium chloride, 50 mM DTT] and 2 μl of 10 mM ATP were added, 10units of T4 polynucleotide kinase was further added, and the reactionwas allowed to proceed at 37° C. for 30 minutes for phosphorylating the5' termini. The, 0.1 μg of the DNA fragment (2.95 kb) derived from theplasmid pBluescript SK(-) and 0.05 μg of the phosphorylated syntheticDNA, each obtained as mentioned above, were added to a total of 20 μl ofsterilized water and ligated to each other using Ready-To-Go T4 DNALigase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNAsolution was used to transform Escherichia coli HB101, and the plasmidpBSES shown in FIG. 88 was obtained. Ten μg of the plasmid obtained wassubjected to sequencing reaction treatment according to the instructionsattached to AutoRead Sequencing Kit (Pharmacia Biotech), followed byelectrophoresis on A.L.F. DNA Sequencer (Pharmacia Biotech) for basesequence determination, whereby it was confirmed that the synthetic DNAhad been introduced as desired.

Then, 3 μg of the plasmid PBSES obtained as mentioned above was added to10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mMsodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA,10 units each of the restriction enzymes EcoRI (Takara Shuzo) and SplI(Takara Shuzo) were further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was fractionated byagarose gel electrophoresis, and about 1 μg of a DNA fragment about 2.95kb in size was recovered.

The, 5 μg of the plasmid pKM796L1 obtained in Paragraph 4 of Example 1was added to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units each of therestriction enzymes EcoRI (Takara Shuzo) and AflIII (Takara Shuzo) werefurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 0.3 μg of an EcoRI-AflIII fragment about 0.39kb in size was recovered.

The, synthetic DNAs respectively having the base sequences shown in SEQID NO:48 and SEQ ID NO:49 were synthesized using an automatic DNAsynthesizer (Applied Biosystems model 380A). To 15 μl of sterilizedwater were added 0.3 μg each of the synthetic DNAs obtained, and themixture was heated at 65° C. for 5 minutes. The reaction mixture wasallowed to stand at room temperature for 30 minutes. Then, 2 μl of10-fold concentrated buffer [500 mM Tris-hydrochloride (pH 7.6), 100 mMmagnesium chloride, 50 mM DTT] and 2 μl of 10 mM ATP were added, 10units of T4 polynucleotide kinase was further added, and the reactionwas allowed to proceed at 37° C. for 30 minutes for phosphorylating the5' termini.

Then, 0.1 μg of the pBSES-derived EcoRI-SplI fragment (2.95 kb), 0.1 μgof the pKM796L1-derived EcoRI-AflIII fragment and 0.05 μg of thephosphorylated synthetic DNA, each obtained as mentioned above, wereadded to a total of 20 μl of sterilized water and ligated together usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pBSL3 shown in FIG. 89 was obtained. Ten μg ofthe plasmid obtained was subjected to sequencing reaction treatmentaccording to the instructions attached to AutoRead Sequencing Kit(Pharmacia Biotech), followed by electrophoresis on A.L.F. DNA Sequencer(Pharmacia Biotech) for base sequence determination, whereby it wasconfirmed that the synthetic DNA had been introduced as desired.

(3) Construction of Human Anti-GM₂ Chimera Antibody Expression VectorpKANTEX796

An human anti-GM₂ chimera antibody expression vector, pKANTEX796, wasconstructed using the plasmid pKANTEX93 obtained in Paragraph 1 ofExample 3 and the plasmids pBSH3 and pBSL3 respectively obtained inParagraph 2 (1) and (2) of Example 3, in the following manner.

Three μg of the plasmid pBSH3 was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of the restriction enzyme ApaI (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was subjected to ethanol precipitation, theprecipitate was dissolved in 10 μl of 50 mM Tris-hydrochloride buffer(pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride, 1mM DTT, 100 μg/ml BSA and 0.01% Triton X-100, 10 units of therestriction enzyme NotI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, and about 0.3μg of an ApaI-NotI fragment about 0.46 kb in size was recovered.

Then, 3 μg of the plasmid pKANTEX93 was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of the restriction enzyme ApaI (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was subjected to ethanol precipitation, theprecipitate was dissolved in 10 μl of 50 mM Tris-hydrochloride buffer(pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride, 1mM DTT, 100 μg/ml BSA and 0.01% Triton X-100, 10 units of therestriction enzyme NotI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, whereby about 1μg of an ApaI-NotI fragment about 12.75 kb in size was recovered.

Then, 0.1 μg of the pBSH3-derived ApaI-NotI fragment and 0.1 μg of thepKANTEX93-derived AnaI-NotI fragment, each obtained as mentioned above,were added to a total of 20 μl of sterilized water and ligated to eachother using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pKANTEX796H shown in FIG. 90 wasobtained.

Then, 3 μg of the plasmid pBSL3 was added to 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 units each of therestriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) werefurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 0.3 μg of an EcoRI-SplI fragment about 0.4 kbin size was recovered.

Then, 3 μg of the plasmid pKANTEX796H was added to 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 units each of therestriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) werefurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 1 μg of an EcoRI-SplI fragment about 13.20 kbin size was recovered.

Then, 0.1 μg of the pBSL3-derived EcoRI-SplI fragment and 0.1 μg of thepKANTEX796H-derived EcoRI-SplI fragment, each obtained as mentionedabove, were added to a total of 20 μl of sterilized water and ligated toeach other using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pKANTEX796 shown in FIG. 91 wasobtained.

(4) Expression of Human Anti-GM₂ Chimera Antibody by pKANTEX796

According to the procedure described in Paragraph 11 of Example 1,pKANTEX796 was introduced into YB2/0 (ATCC CRL 1581) cells and, as aresult of selection by means of G418 (0.5 mg/ml) and MTX (200 nM), acell line capable of producing about 1 to 2 μg/ml of human anti-GM₂chimera antibody was obtained. It was confirmed that efficient andstable humanized antibody expression is possible using expression vectorpKANTEX93.

3. Transient Humanized Antibody Expression in COS-7 (ATCC CRL 1651)Cells

For enabling more rapid activity evaluation of various versions of humanCDR-grafted anti-GM₂ antibody, transient expression of human anti-GM₂chimera antibody expression was caused in COS-7 cells by theLipofectamine method using pKANTEX796 and a variant thereof in thefollowing manner.

(1) Construction of Variant of pKANTEX796

Since transient antibody expression in animal cells is dependent on thecopy number of an expression vector introduced, it was supposed that anexpression vector smaller in size would show a higher expressionefficiency. Therefore, a smaller humanized antibody expression vector,pT796, was constructed by deleting a region supposedly having no effecton humanized antibody expression from pKANTEX796 in the followingmanner.

Thus, 3 μg of the plasmid pKANTEX796 was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 50 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzymeHindIII (Takara Shuzo) was further added, and the reaction was allowedto proceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, the precipitate was dissolved in 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, 10 units of the restriction enzymeMluI (Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation, and the 5' cohesive ends resulting from therestriction enzyme digestion were rendered blunt using DNA Blunting Kit(Takara Shuzo). The reaction mixture was fractionated by agarose gelelectrophoresis and about 1 μg of a DNA fragment about 9.60 kb in sizewas recovered. A 0.1-μg portion of the thus-recovered DNA fragment wasadded to a total of 20 μl of sterilized water and subjected to ligationtreatment using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Thethus-obtained recombinant plasmid DNA solution was used to transformEscherichia coli HB101, and the plasmid pT796 shown in FIG. 92 wasobtained.

(2) Transient Expression of Human Anti-GM₂ Chimera Antibody UsingpKANTEX796 and pT796

A 1×10⁵ cells/ml suspension of COS-7 cells was distributed in 2-mlportions into wells of a 6-well plate (Falcon) and cultured overnight at37° C. Two μg of pKANTEX796 or pT796 was added to 100 μl of OPTI-MEMmedium (Gibco), a solution prepared by adding 10 μl of LIPOFECTAMINEreagent (Gibco) to 100 μl of OPTI-MEM medium (Gibco) was further added,and the reaction was allowed to proceed at room temperature for 40minutes to cause DNA-liposome complex formation. The COS-7 cellscultured overnight were washed twice with 2 ml of OPTI-MEM medium(Gibco), the complex-containing solution was added, and the cells werecultured at 37° C. for 7 hours. Then, the solution was removed, 2 ml ofDMEM medium (Gibco) containing 10% FCS was added to each well, and thecells were cultured at 37° C. After 24 hours, 48 hours, 72 hours, 96hours and 120 hours of cultivation, the culture supernatant wasrecovered and, after concentration procedure as necessary, evaluated forhuman anti-GM₂ chimera antibody activity in the culture supernatant bythe ELISA method described in Paragraph 11 of Example 1. The results areshown in FIG. 93. As shown in FIG. 93, higher levels of transient humananti-GM₂ chimera antibody expression was observed with pT796 as comparedwith pKANTEX796. For pT796, the level of expression was highest at 72 to96 hours, the concentration being about 30 ng/ml (in terms of GM₂binding activity). The above results indicate that construction of apKANTEX93-derived vector having a reduced size and introduction thereofinto COS-7 cells make it possible to make activity evaluation ofexpression vector-derived humanized antibodies in a transient expressionsystem. Furthermore, for close activity comparison of various versionsof human CDR-grafted anti-GM₂ antibody as mentioned hereinafter, theELISA method described below under (3) was used to determine antibodyconcentrations in transient expression culture supernatants.

(3) Determination by ELISA of Humanized Antibody Concentrations inTransient Expression Culture Supernatants

A solution prepared by 400-fold dilution of goat anti-human IgG (γchain) antibody (Igaku Seibutugaku Kenkyusho) with PBS was distributedin 50-μl portions into wells of a 96-well microtiter plate and allowedto stand overnight at 4° C. for binding to the wells. After removing theantibody solution, blocking was effected with 100 μl of PBS containing1% BSA at 37° C. for 1 hour. Fifty μl of a transient expression culturesupernatant or purified human anti-GM₂ chimera antibody was addedthereto and allowed to react at room temperature for 1 hour. Thereafter,the solution was removed, the wells were washed with PBS, and 50 μl of asolution prepared by 500-fold dilution of peroxidase-labeled mouseanti-human _(K) L chain antibody (Zymet) with PBS was added and allowedto react at room temperature for 1 hour. After washing with PBS, 50 μlof an ABTS substrate solution [prepared by dissolving 550 mg of2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in0.1 M citrate buffer (pH 4.2) and adding, just before use, 1 μl/ml ofhydrogen peroxide] was added for causing color development, and theOD₄₁₅ was measured.

4. Production of Human CDR-Grafted Anti-GM₂ Antibody Using HumanizedAntibody Expression Vector pKANTEX93

A human CDR-grafted anti-GM₂ antibody higher in GM₂ -binding activitythan the human CDR-grafted anti-GM₂ antibody described in Example 2 wasproduced in the following manner.

(1) Modification of Human CDR-Grafted Anti-GM₂ Antibody H Chain VariableRegion Described in Paragraph 1 (1) of Example 2

DNAs coding for some versions of the human CDR-grafted anti-GM₂ antibodyH chain variable region described in Example 2 as derived by replacingseveral amino acids in regions other than the CDR (framework;hereinafter referred to as FR) with original mouse antibody amino acidswere constructed in the following manner. Based on a computer model forthe variable region of mouse KM796, those amino acid residues that wereexpected to contribute to restoration of antigen-binding activity as aresult of mutation were selected as the amino acid residues to bemutated.

First, DNAs respectively having the base sequences of SEQ ID NO:50 andSEQ ID NO:51 were synthesized using an automatic DNA synthesize (AppliedBiosystems model 380A).

Then, a version (version 2) of human CDR-grafted antibody H chainvariable region shown in SEQ ID NO:52 and having mutation in positions78 (threonine in lieu of glutamine), 79 (alanine in lieu ofphenylalanine) and 80 (tyrosine in lieu of serine) was constructed inthe same manner as in Paragraph 1 (1) of Example 2 using a synthetic DNAof SEQ ID NO:50 in lieu of the synthetic DNA of SEQ ID NO:27.

Then, another version (version 4) of human CDR-grafted antibody H chainvariable region shown in SEQ ID NO:53 and having mutations in positions27 (tyrosine in lieu of phenylalanine), 30 (threonine in lieu ofserine), 40 (serine in lieu of proline) and 41 (histidine in lieu ofproline) was constructed in the same manner as in Paragraph 1 (1) ofExample 2 using a synthetic DNA of SEQ ID NO:51 in lieu of the syntheticDNA of SEQ ID NO:25.

(2) Construction of Human CDR-Grafted Anti-GM₂ Antibody H Chain VariableRegion Using Known Common Human Antibody H Chain Variable Region

According to Kabat et al. (Kabat E. A. et al., "Sequences of Proteins ofImmunological Interest", US Dept. of Health and Human Services, 1991),known human antibody H chain variable regions are classifiable intosubgroups I to III (HSG I to III) based on the homology of their FRregions, and common sequences have been identified for respectivesubgroups. Therefore, a human CDR-grafted anti-GM₂ antibody H chainvariable region was constructed based on those common sequences. First,for selecting common sequences to serve as the base, the homology wasexamined between the FR of the mouse KM796 H chain variable region andthe common sequence FR of the human antibody H chain variable region ofeach subgroup (Table 3).

Table 3

Homology (%) between mouse KM796 H chain variable region FR and humanantibody H chain variable region common sequence FR

    ______________________________________                                        HSG I           HSG II  HSG III                                               ______________________________________                                        72.1            52.9    58.6                                                  ______________________________________                                    

As a result, it was confirmed that subgroup I shows the greatestsimilarity. Thus, based on the common sequences of subgroup I, a humanCDR-grafted anti-GM₂ antibody H chain variable region was constructed bythe PCR method in the following manner.

Synthetic DNAs respectively having the base sequences of SEQ ID NO:54through SEQ ID NO:59 were synthesized using an automatic DNA synthesizer(Applied Systems model 380A). The DNAs synthesized were added, each to afinal concentration of 0.1 μM, to 50 μl of 10 mM Tris-hydrochloridebuffer (pH 8.3) containing 50 mM potassium chloride, 1.5 mM magnesiumchloride, 0.001% gelatin, 200 μM dNTP, 0.5 μM M13 primer RV (TakaraShuzo), 0.5 μM M13 primer M4 (Takara Shuzo) and 2 units of TaKaRa TaqDNA polymerase, the mixture was covered with 50 μl of mineral oil, a DNAthermal cycler (Perkin Elmer model PJ480) was loaded with the mixture,and 30 PCR cycles (2 minutes at 94° C., 2 minutes at 55° C. and 2minutes at 72° C. per cycle) were conducted. The reaction mixture waspurified using QIAquick PCR Purification Kit (Qiagen) and then made intoa solution in 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme ApaI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasadded to 10 μl of 50 mM Tris-hydrochloride (pH 7.5) containing 100 mMsodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100 μg/ml BSA and0.01% Triton X-100, 10 units of the restriction enzyme NotI (TakaraShuzo) was further added, and the reaction was allowed to proceed at 37°C. for 1 hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 0.2 μg of an ApaI-NotI fragment about 0.44 kbin size was recovered.

Then, 3 μg of the plasmid pBSH3 obtained in Paragraph 2 (1) of Example 3was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme ApaI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasadded to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100 μg/mlBSA and 0.01% Triton X-100, 10 units of the restriction enzyme NotI(Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was fractionated byagrose gel electrophoresis, and about 1 μg of an ApaI-NotI fragmentabout 2.95 kb in size was recovered.

Then, 0.1 μg of the ApaI-NotI fragment of the human CDR-grafted anti-GM₂antibody H chain variable region and 0.1 μg of the ApaI-NotI fragment ofpBSH3, each obtained as mentioned above, were added to a total of 20 μlof sterilized water and ligated to each other using Ready-To-Go T4 DNALigase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNAsolution was used to transform Escherichia coli HB101. Plasmid DNAs wereprepared from 10 transformant clones and their base sequences weredetermined. As a result, a plasmid, pBSH10, shown in FIG. 94 and havingthe desired base sequence was obtained. The amino acid sequence and basesequence of the human CDR-grafted anti-GM₂ antibody H chain variableregion contained in pBSH10 are shown in SEQ ID NO:60. In the amino acidsequence of the thus-constructed human CDR-grafted anti-GM₂ antibody Hchain variable region, arginine in position 67, alanine in position 72,serine in position 84 and arginine in position 98 in the FR as selectedbased on a computer model for the variable region are replaced withlysine, valine, histidine and threonine, respectively, that are found inthe mouse KM796 H chain variable region. This is for the purpose ofretaining the antigen-binding capacity of mouse KM796.

(3) Modification of Human CDR-Grafted Anti-GM₂ Antibody L Chain VariableRegion Described in Paragraph 1 (2) of Example 2

First, a DNA having the base sequence of SEQ ID NO:61 was synthesizedusing an automatic DNA synthesizer (Applied Biosystems model 380A), anda human CDR-grafted anti-GM₂ antibody L chain variable region cDNA witha 3' terminus capable of pairing with the restriction enzyme SplI wasconstructed by following the same reaction procedure as in Paragraph 1(2) of Example 2 using the synthetic DNA in lieu of the synthetic DNA ofSEQ ID NO:35.

Then, 3 μg of the plasmid pBSL3 obtained in Paragraph 2 (2) of Example 3was added to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTTand 100 μg/ml BSA, 10 units each of the restriction enzymes EcoRI(Takara Shuzo) and SplI (Takara Shuzo) were further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, and about 1 μgof an EcoRI-SplI fragment about 2.95 kb in size was recovered.

Then, 0.1 μg of the EcoRI-SplI fragment of the human CDR-graftedanti-GM₂ antibody L chain variable region obtained as mentioned aboveand 0.1 μg of the above EcoRI-SplI fragment of pBSL3 were added to atotal of 20 μl of sterilized water and ligated to each other usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pBSL16 shown in FIG. 95 was obtained.

Then, DNAs coding for certain versions of the human CDR-grafted anti-GM₂antibody L chain variable region contained in the above plasmid pBSL16were constructed by replacing a certain number of amino acids in the FRwith original mouse antibody amino acids by mutagenesis by means of PCRin the following manner (FIG. 96). Based on a computer model for thevariable region of mouse KM796, those amino acid residues that wereexpected to contribute to restoration of antigen-binding activity as aresult of mutation were selected as the amino acid residues to bemutated.

Antisense and sense DNA primers for introducing mutations weresynthesized using an automatic DNA synthesizer (Applied Biosystems model380A). A first PCR reaction was conducted in the same manner as inParagraph 4 (2) of Example 3 using a final concentration each of 0.5 μMof M13 primer RV (Takara Shuzo) and the antisense DNA primer and of M13primer M4 (Takara Shuzo) and the sense DNA primer, with 1 ng of pBSL16as the template. Each reaction mixture was purified using QIAquick PCRPurification Kit (Qiagen) with elution with 20 μl of 10 mMTris-hydrochloride (pH 8.0). Using 5 μl of each eluate, a second PCRreaction was conducted in the same manner as in Paragraph 4 (2) ofExample 3. The reaction mixture was purified using QIAaquick PCRPurification Kit (Qiagen) and then made into a solution in 30 μl of 50mM Tris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride,10 mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 units each ofthe restriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo)were further added, and the reaction was allowed to proceed at 37° C.for 1 hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 0.2 μg of an EcoRI-SplI fragment (about 0.39kb) of each mutant version of the human CDR-grafted anti-GM₂ antibody Lchain variable region was recovered.

Then, 0.1 μg of the above EcoRI-SplI fragment of each mutant version ofthe human CDR-grafted anti-GM₂ antibody L chain variable region and 0.1μg of the EcoRI-SplI fragment of pBSL3 were added to a total of 20 μl ofsterilized water and ligated to each other using Ready-To-Go T4 DNAligase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNAsolution was used to transform Escherichia coli HB101, and a plasmid DNAwas prepared from a transformant clone, and the base sequence of saidplasmid was determined. In this way, plasmids respectively containing abase sequence having a desired mutation or mutations were obtained.

Thus, a plasmid, pBSLV1, containing version 1, shown in SEQ ID NO:64, ofthe human CDR-grafted anti-GM₂ antibody L chain variable region wasobtained following the above procedure using the synthetic DNA of SEQ IDNO:62 as the mutant antisense primer and the synthetic DNA of SEQ IDNO:63 as the mutant sense primer. In the amino acid sequence of theversion 1 human CDR-grafted anti-GM₂ antibody L chain variable region,the amino acid valine in position 15 in the FR is replaced with prolinethat is found in the mouse KM796 L chain variable region. This is forthe purpose of retaining the antigen-binding capacity of mouse KM796.

A plasmid, pBSLV2, containing version 2, shown in SEQ ID NO:67, of thehuman CDR-grafted anti-GM₂ antibody L chain variable region was obtainedfollowing the above procedure using the synthetic DNA of SEQ ID NO:65 asthe mutant antisense primer and the synthetic DNA of SEQ ID NO:66 as themutant sense primer. In the amino acid sequence of the version 2 humanCDR-grafted anti-GM₂ antibody L chain variable region, the amino acidleucine in positions 46 in the FR is replaced with tryptophan that isfound in the mouse KM796 L chain variable region. This is for thepurpose of retaining the antigen-binding capacity of mouse KM796.

A plasmid, pBSLV3, containing version 3, shown in SEQ ID NO:70, of thehuman CDR-grafted anti-GM₂ antibody L chain variable region was obtainedfollowing the above procedure using the synthetic DNA of SEQ ID NO:68 asthe mutant antisense primer and the synthetic DNA of SEQ ID NO:69 as themutant sense primer. In the amino acid sequence of the version 3 humanCDR-grafted anti-GM₂ antibody L chain variable region, proline inposition 79 and isoleucine in position 82 in the FR are both replacedwith alanine that is found in the mouse KM796 L chain variable region.This is for the purpose of retaining the antigen-binding capacity ofmouse KM796.

Then, a plasmid, pBSLV1+2, containing a human CDR-grafted anti-GM₂antibody L chain variable region having both the version 1 and version 2mutations was constructed in the following manner.

Three μg of the plasmid pBSLVl obtained as mentioned above was added to10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50 mMsodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units each ofthe restriction enzymes EcoRI (Takara Shuzo) and HindIII (Takara Shuzo)were further added, and the reaction was allowed to proceed at 37° C.for 1 hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 0.2 μg of an EcoRI-HindIII fragment about0.20 kb in size was recovered.

Then, 3 μg of the plasmid pBSLV2 obtained as mentioned above was addedto 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50 mMsodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units each ofthe restriction enzymes EcoRI (Takara Shuzo) and HindIII (Takara Shuzo)were further added, and the reaction was allowed to proceed at 37° C.for 1 hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 1 μg of an EcoRI-HindIII fragment about 3.2kb in size was recovered.

Then, 0.1 μg of the EcoRI-HindIII fragment of pBSLV1 and 0.1 μg of theEcoRI-HindIII fragment of pBSLV2, each obtained as mentioned above, wereadded to a total of 20 μl of sterilized water and ligated to each otherusing Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pBSLV1+2 shown in FIG. 97 was obtained.

Then, the PCR reaction procedure mentioned above was followed using 1 ngof the plasmid pBSLV1+2 obtained as mentioned above as the template, asynthetic DNA having the base sequence of SEQ ID NO:71 as the mutantantisense primer and a synthetic DNA having the base sequence of SEQ IDNO:72 as the mutant sense primer, whereby a plasmid, pBSLV4, containinga version 4 human CDR-grafted anti-GM₂ antibody L chain variable regionset forth in SEQ ID NO:73 was obtained. In the amino acid sequence ofthe version 4 human CDR-grafted anti-GM₂ antibody L chain variableregion, valine in position 15, leucine in position 46, aspartic acid inposition 69, phenylalanine in position 70 and threonine in position 71in the FR are replaced with proline, tryptophan, serine, tyrosine andserine, respectively, that are found in the mouse KM796 L chain variableregion. This is for the purpose of retaining the antigen-bindingcapacity of mouse KM796.

Then, the PCR reaction procedure mentioned above was followed using 1 ngof the plasmid pBSLV1+2 obtained as mentioned above as the template, asynthetic DNA having the base sequence of SEQ ID NO:74 as the mutantantisense primer and a synthetic DNA having the base sequence of SEQ IDNO:75 as the mutant sense primer, whereby a plasmid, pBSLV8, containinga version 8 human CDR-grafted anti-GM₂ antibody L chain variable regionset forth in SEQ ID NO:76 was obtained. In the amino acid sequence ofthe version 8 human CDR-grafted anti-GM₂ antibody L chain variableregion, valine in position 15, leucine in position 46, aspartic acid inposition 69, phenylalanine in position 70, threonine in position 71,serine in position 76, leucine in position 77 and glutamine in position78 in the FR are replaced with proline, tryptophan, serine, tyrosine,serine, arginine, methionine and glutamic acid, respectively, that arefound in the mouse KM796 L chain variable region. This is for thepurpose of retaining the antigen-binding capacity of mouse KM796.

Then, the PCR reaction procedure mentioned above was followed using 1 ngof the plasmid pBSLV4 obtained as mentioned above as the template, asynthetic DNA having the base sequence of SEQ ID NO:77 as the mutantantisense primer and a synthetic DNA having the base sequence of SEQ IDNO:78 as the mutant sense primer, whereby a plasmid, pBSLm-2, containinga version Lm-2 human CDR-grafted anti-GM₂ antibody L chain variableregion set forth in SEQ ID NO:79 was obtained. In the amino acidsequence of the version Lm-2 human CDR-grafted anti-GM₂ antibody L chainvariable region, valine in position 15, tyrosine in position 35, leucinein position 46, aspartic acid in position 69, phenylalanine in position70 and threonine in position 71 in the FR are replaced with proline,phenylalanine, tryptophan, serine, tyrosine and serine, respectively,that are found in the mouse KM796 L chain variable region. This is forthe purpose of retaining the antigen-binding capacity of mouse KM796.

Then, the PCR reaction procedure mentioned above was followed using 1 ngof the plasmid pBSLV4 obtained as mentioned above as the template, asynthetic DNA having the base sequence of SEQ ID NO:80 as the mutantantisense primer and a synthetic DNA having the base sequence of SEQ IDNO:81 as the mutant sense primer, whereby a plasmid, pBSLm-8, containinga version Lm-8 human CDR-grafted anti-GM₂ antibody L chain variableregion set forth in SEQ ID NO:82 was obtained. In the amino acidsequence of the version Lm-8 human CDR-grafted anti-GM₂ antibody L chainvariable region, valine in position 15, leucine in posit8ion 46,aspartic acid in position 69, phenylalanine in position 70, threonine inposition 71, phenylalanine in position 72 and serine in position 76 inthe FR are replaced with proline, tryptophan, serine, tyrosine, serine,leucine and arginine, respectively, that are found in the mouse KM796 Lchain variable region. This is for the purpose of retaining theantigen-binding capacity of mouse KM796.

Then, a plasmid, pBSLm-28, containing a human CDR-grafted anti-GM₂antibody L chain variable region having both the version Lm-2 andversion Lm-8 mutations was constructed in the following manner.

Three μg of the plasmid pBSLm-2 obtained as mentioned above was added to10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mMsodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme EcoRI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasadded to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/mlBSA, 10 units of the restriction enzyme XbaI (Takara Shuzo) was furtheradded, and the reaction as allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis, andabout 0.2 μg of an EcoRI-XbaI fragment about 0.24 kb in size wasrecovered.

Then, 3 μg of the plasmid pBSLm-8 obtained as mentioned above was addedto 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 100 mMsodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme EcoRI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasadded to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 50mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/mlBSA, 10 units of the restriction enzyme XbaI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis, andabout 1 μg of an EcoRI-XbaI fragment about 3.16 kb in size wasrecovered.

Then, 0.1 μg of the EcoRI-XbaI fragment of pBSLm-2 and 0.1 μg of theEcoRI-XbaI fragment of pBSLm-8, each obtained as mentioned above, wereadded to a total of 20 μl of sterilized water and ligated to each otherusing Ready-To-go T4 DNA Ligase (Pharmacia Biotech). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pBSLm-28 shown in FIG. 98 was obtained. Theversion Lm-28 human CDR-grafted anti-GM₂ antibody L chain variableregion contained in the plasmid pBSLm-28 is shown in SEQ ID NO:83. Inthe amino acid sequence of the version Lm-28 human CDR-grafted anti-GM₂antibody L chain variable region thus constructed, valine in position15, tyrosine in position 35, leucine in position 46, aspartic acid inposition 69, phenylalanine in position 70, threonine in position 71,phenylalanine in position 72 and serine in position 76 are replaced withproline, phenylalanine, tryptophan, serine, tyrosine, serine, leucineand arginine, respectively, that are found in the mouse KM796 L chainvariable region. This is for the intended purpose of retaining theantigen-binding capacity of mouse KM796.

(4) Construction of Human CDR-Grafted Anti-GM₂ Antibody L Chain VariableRegion Using Known Common Human Antibody L Chain Variable Region

According to Kabat et al. (Kabat E. A. et al., "Sequences of Proteins ofImmunological Interest", US Dept. of Health and Human Services, 1991),known human antibody L chain variable regions are classifiable intosubgroups I to IV based on the homology of their FR regions, and commonsequences have been identified for respective subgroups. Therefore, ahuman CDR-grafted anti-GM₂ antibody L chain variable region wasconstructed based on those common sequences. First, for selecting commonsequences to serve as the base, the homology was examined between the FRof the mouse KM796 L chain variable region and the common sequence FR ofthe human antibody L chain variable region of each subgroup (Table 4).

    ______________________________________                                        HSG I    HSG II        HSG III HSG IV                                         ______________________________________                                        70.0     65.0          68.8    67.5                                           ______________________________________                                    

As a result, it was confirmed that subgroup I shows the greatestsimilarity. Thus based on the common sequence of subgroup I, a humanCDR-grafted anti-GM₂ antibody L chain variable region was constructed bythe PCR method in the following manner.

Synthetic DNAs respectively having the base sequences of SEQ ID NO:84through SEQ ID NO:89 were synthesized using an automatic DNA synthesizer(Applied Systems model 380A). The DNAs synthesized were added, each to afinal concentration of 0.1 μM, to 50 μl of 10 mM Tris-hydrochloridebuffer (pH 8.3) containing 50 mM potassium chloride, 1.5 mM magnesiumchloride, 0.001% gelatin, 200 μM dNTP, 0.5 μM M13 primer RV (TakaraShuzo), 0.5 μM M13 primer M4 (Takara Shuzo) and 2 units of TaKaRa TaqDNA polymerase. The mixture was covered with 50 μl of mineral oil, a DNAthermal cycler (Perkin Elmer model PJ480) was loaded with the mixture,and 30 PCR cycles (2 minutes at 94° C., 2 minutes at 55° C. and 2minutes at 72° C. per cycle) were conducted. The reaction mixture waspurified using QIAquick PCR Purification Kit (Qiagen) and then made intoa solution in 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTTand 100 μg/ml BSA, 10 units each of the restriction enzymes EcoRI(Takara Shuzo) and SplI (Takara Shuzo) were further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, and about 0.2μg of an EcoRI-SplI fragment about 0.39 kb in size was recovered.

Then, 0.1 μg of the above ECoRI-SplI fragment of the human CDR-graftedanti-GM₂ antibody L chain variable region and 0.1 μg of the EcoRI-SplIfragment of pBSL3 were added to a total of 20 μl of sterilized water andligated to each other using Ready-To-Go T4 DNA Ligase (PharmaciaBiotech). The thus-obtained recombinant plasmid DNA solution was used totransform Escherichia coli HB101. Plasmid DNAs were prepared from 10transformant clones and their base sequences were determined. As aresult, a plasmid, PBSHSGL, shown in FIG. 99 and having the desired basesequence was obtained. The amino acid sequence and base sequence of thehuman CDR-grafted anti-GM₂ antibody L chain variable region contained inpBSHSGL are shown in SEQ ID NO:90. In the amino acid sequence of thethus-constructed human CDR-grafted anti-GM₂ antibody L chain variableregion, methionine in position 4, leucine in position 11, valine inposition 15, tyrosine in position 35, alanine in position 42, leucine inposition 46, aspartic acid in position 69, phenylalanine in position 70,threonine in position 71, leucine in position 77 and valine in position103 in the FR as selected based on a computer model for the variableregion are replaced with leucine, methionine, proline, phenylalanine,serine, tryptophan, serine, tyrosine, serine, methionine and leucine,respectively, that are found in the mouse KM796 L chain variable region.This is for the intended purpose of retaining the antigen-bindingcapacity of mouse MK796.

(5) Activity Evaluation of Mutant Versions of Human CDR-Grafted Anti-GM₂Antibody in Terms of Transient Expression

Various mutant version human CDR-grafted anti-GM₂ antibodies composed ofthe human CDR-grafted anti-GM₂ antibody H chain and L chain variableregions constructed in Paragraphs (1) through (4) of Example 3 andhaving varying mutations were evaluated for activity in terms oftransient expression in the following manner.

First, for evaluating the human CDR-grafted anti-GM₂ antibody H chainvariable regions having varying mutations, expression vectors,pT796HCDRHV2, pT796HCDRHV4 and pT796HCDRH10, were constructed byreplacing the mouse H chain variable region of the human anti-GM₂chimera antibody transient expression vector pT796 obtained in Paragraph3 (1) of Example 3 with the human CDR-grafted anti-GM₂ antibody H chainvariable regions having varying mutations, in the following manner. Forcomparison, an expression vector, pT796HCDR was constructed by replacingthe mouse H chain variable region of pT796 with the human CDR-graftedanti-GM₂ antibody H chain variable region obtained in Paragraph 1 (1) ofExample 1.

Three μg of the plasmid pT796 was added to 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 units each of therestriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) werefurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 1 μg of an EcoRI-SplI fragment about 9.20 kbin size was recovered.

Then, 3 μg of the plasmid pBSL16 obtained in Paragraph 4 (3) of Example3 was added to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTTand 100 μg/ml BSA, 10 units each of the restriction enzymes EcoRI(Takara Shuzo) and SplI (Takara Shuzo) were further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, and about 0.3μg of an EcoRI-SplI fragment about 0.39 kb in size was recovered.

Then, 0.1 μg of the EcoRI-SplI fragment of pT796 and 0.1 μg of theEcoRI-SplI fragment of pBSL16, each obtained as mentioned above, wereadded to a total of 20 μl of sterilized water and ligated to each otherusing Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). The thus-obtainedrecombinant plasmid DNA solution was used to transform Escherichia coliHB101, and the plasmid pT796LCDR shown in FIG. 100 was obtained.

Then, 3 μg of the above plasmid pT796LCDR was added to 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of the restriction enzyme ApaI (Takara Shuzo) wasfurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was subjected to ethanol precipitation, theprecipitate was added to 10 μl of 50 mM Tris-hydrochloride buffer (pH7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mMDTT, 100 μg/ml BSA and 0.01% Triton X-100, 10 units of the restrictionenzyme NotI (Takara Shuzo) was further added, and the reaction wasallowed to proceed at 37° C. for 1 hour. The reaction mixture wasfractionated by agarose gel electrophoresis, and about 1 μg of anApaI-NotI fragment about 9.11 kb in size was recovered.

Then, 0.1 μg of the human CDR-grafted anti-GM₂ antibody H chain variableregion obtained in Paragraph 1 (1) of Example 2 or the mutant version 2or 4 human CDR-grafted anti-GM₂ antibody H chain variable regionobtained in Paragraph 4 (1) of Example 3 and 0.1 μg of the ApaI-NotIfragment of pT796LCDR were added to a total of 20 μl of sterilized waterand ligated to each other using Ready-To-Go T4 DNA Ligase (PharmaciaBiotech). Each recombinant plasmid DNA solution thus obtained was usedto transform Escherichia coli HB101. The plasmids pT796HLCDR,pT796HLCDRHV2 and pT796HLCDRHV4 shown in FIG. 101 were obtained.

Then, 3 μg of the plasmid pBSH10 obtained in Paragraph 4 (2) of Example3 was added to 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of therestriction enzyme ApaI (Takara Shuzo) was further added, and therestriction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was subjected to ethanol precipitation, the precipitate wasadded to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100 μg/mlBSA and 0.01% Triton X-100, 10 units of the restriction enzyme NotI(Takara Shuzo) was further added, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was fractionated byagarose gel electrophoresis, and about 0.3 μg of an ApaI-NotI fragmentabout 0.44 kb in size was recovered.

Then, 0.1 μg of the ApaI-NotI fragment of pBSM10 and 0.1 μg of theApaI-NotI fragment of pT796LCDR were added to a total of 20 μl ofsterilized water and ligated to each other using Ready-To-Go T4 DNALigase (Pharmacia Biotech). The thus-obtained recombinant plasmid DNAsolution was used to transform Escherichia coli HB101, and the plasmidpT796HLCDRH10 shown in FIG. 102 was obtained.

Then, 3 μg each of the plasmids pT796HLCDR, pT796HLCDRHV2, pT796HLCDRHV4and pT796HLCDRH10 were respectively added to 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 units each of therestriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) werefurther added, and the reaction was allowed to proceed at 37° C. for 1hour. Each reaction mixture was fractionated by agarose gelelectrophoresis, and about 1 μg of an EcoRI-SplI fragment about 9.15 kbin size was recovered.

Then, 5 μg of the plasmid pBSL3 obtained in Paragraph 2 (2) of Example 3was added to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTTand 100 μg/ml BSA, 10 units each of the restriction enzymes EcoRI(Takara Shuzo) and SplI (Takara Shuzo) were further added, and thereaction was allowed to proceed at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, and about 0.4μg of an EcoRI-SplI fragment about 0.39 kb in size was recovered.

Then, 0.1 μg of the EcoRI-SplI fragment of each of pT796HLCDR,pT796HLCDRHV2, pT796HLCDRHV4 and pT796HLCDRH10 and 0.1 μg of theEcoRI-SplI fragment of pBSL3 were added to a total of 20 μl ofsterilized water and ligated to each other using Ready-To-Go DNA Ligase(Pharmacia Biotech). Each recombinant plasmid DNA solution thus obtainedwas used to transform Escherichia coli HB101. In this way, the plasmidspT796HCDR, pT796HCDRHV2, pT796HCDRHV4 and pT796HCDRH10 shown in FIG. 103were obtained.

Then, 2 μg each of the plasmids pT796HCDR, pT796HCDRHV2, pT796HCDRHV4and pT796HCDRH10 thus obtained were used for transient human CDR-graftedanti-GM₂ antibody expression and for culture supernatant humanCDR-grafted anti-GM₂ antibody activity evaluation by the proceduresdescribed in Paragraph 3 (2) and (3) of Example 3, respectively. Afterintroduction of each plasmid, the culture supernatant was recovered at72 hours, and the GM₂ -binding activity and antibody concentration inthe culture supernatant were determined by ELISA and the relativeactivity was calculated with the activity of the positive controlchimera antibody taken as 100%. The results are shown in FIG. 104.

The results revealed that the amino acid mutations alone in mutantversions 2 and 4 have little influence on the restoration of theantigen-binding activity of the human CDR-grafted anti-GM₂ antibody butthat the use of the pBSH10-derived human CDR-grafted antibody H chainvariable region constructed based on the known human antibody H chainvariable region common sequence, contributes to the restoration of theantigen-binding activity.

In view of the above results, the human CDR-grafted anti-GM₂ antibody Hchain variable region constructed based on the known human antibody Hchain variable region common sequence as shown in SEQ ID NO:60 wasselected as a novel human CDR-grafted anti-GM₂ antibody H chain variableregion.

Then, for evaluating the human CDR-grafted anti-GM₂ antibody L chainvariable regions having varying variations, expression vectors,pT796HLCDRLVl, pT796HLCDRLV2, pT796HLCDRLV3, pT796HLCDRLV4,pT796HLCDRLV8, pT796HLCDRLm-2, pT796HLCDRLm-8, pT796HLCDRLm-28 andpT796HLCDRHSGL, were constructed in the following manner by replacingthe mouse L chain variable region of the vector pT796HCDRH10 fortransient human CDR-grafted anti-GM₂ antibody expression obtained asmentioned above with the human CDR-grafted anti-GM₂ antibody L chainvariable regions having varying mutations.

Thus, 3 μg of the plasmid pT796HCDRH10 was added to 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 units each of therestriction enzymes EcoRI (Takara Shuzo) and SplI (Takara Shuzo) werefurther added, and the reaction was allowed to proceed at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectro-phoresis, and about 1 μg of an EcoRI-SplI fragment about 9.15 kbin size was recovered.

Then, 3 μg of the plasmid pBSLV1, pBSLV2, pBSLV3, pBSLV4, pBSLV8,pBSLm-2, pBSLm-8, pBSLm-28 or PBSHSGL obtained in Paragraph (3) or (4)of Example 3 was added to 10 μl of 50 mM Tris-hydrochloride buffer (pH7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mMDTT and 100 μg/ml BSA, 10 units each of the restriction enzymes EcoRI(Takara Shuzo) and SplI (Takara Shuzo) were further added, and thereaction was allowed to proceed at 37° C. for 1 hour. Each reactionmixture was fractionated by agarose gel electrophoresis, and about 0.3μg of an EcoRI-SplI fragment about 0.39 kb in size was recovered.

Then, 0.1 μg of the EcoRI-SplI fragment of the pT796HCDRH10 and 0.1 μgof the EcoRI-SplI fragment of each mutant version human CDR-graftedanti-GM₂ antibody L chain variable region were added to a total of 20 μlof sterilized water and ligated to each other using Ready-To-Go T4 DNALigase (Pharmacia Biotech). Each recombinant plasmid DNA solution thusobtained was used to transform Escherichia coli HB101. In this way, theplasmids pT796HLCDRLV1, pT796HLCDRLV2, pT796HLCDRLV3, pT796HLCDRLV4,pT796HLCDRLV8, pT796HLCDRLm-2, pT796HLCDRLm-8, pT796HLCDRLm-28 andpT796HLCDRHSGL were obtained as shown in FIG. 105.

Then, 2 μg each of the thus-obtained plasmids pT796HLCDRLV1,pT796HLCDRLV2, pT796HLCDRLV3, pT796HLCDRLV4, pT796HLCDRLV8,pT796HLCDRLm-2, pT796HLCDRLm-8, pT796HLCDRLm-28 and pT796HLCDRHSGL andof the plasmid pT796HLCDR described in Example 2 and capable ofexpressing human CDR-grafted anti-GM₂ antibody were used for transienthuman CDR-grafted anti-GM₂ antibody expression and for culturesupernatant human CDR-grafted anti-GM₂ antibody activity evaluation bythe procedures described in Paragraph 3 (2) and (3) of Example 3,respectively. After introduction of each plasmid, the culturesupernatant was recovered at 72 hours, and the GM₂ -binding activity andantibody concentration in the culture supernatant were determined byELISA and the relative activity was calculated with the activity of thepositive control chimera antibody taken as 100%. The results are shownin FIG. 106.

The results revealed that the amino acid mutations alone in mutantversions 1, 2, 3, 4 and 8 have little influence on the restoration ofthe antigen-binding activity of the human CDR-grafted anti-GM₂ antibodybut that the amino acid mutations in mutant versions Lm-2 and Lm-8contributes to the restoration of the antigen-binding activity.Furthermore, version Lm-28 having both the amino acid mutations of Lm-2and Lm-8 showed a high level of antigen-biding activity almostcomparable to that of the chimera antibody, revealing that those aminoacids mutated in producing Lm-28 were very important from theantigen-binding activity viewpoint.

In view of the above results, the version Lm-28 human CDR-graftedanti-GM₂ antibody L chain variable region shown in SEQ ID NO:83 wasselected as a first novel human CDR-grafted anti-GM₂ antibody L chainvariable region.

It was further revealed that the antigen-binding activity can berestored when the pBSHSGL-derived human CDR-grafted anti-GM₂ antibody Lchain variable region, namely the human CDR-grafted anti-GM₂ antibody Lchain variable region constructed based on the known human antibody Lchain variable region common sequence, is used.

In view of the above result, the human CDR-grafted anti-GM₂ antibody Lchain variable region constructed based on the known human antibody Lchain variable region common sequence as set forth in SEQ ID NO:90 wasselected as a second novel human CDR-grafted anti-GM₂ antibody L chainvariable region.

It is to be noted that in those human CDR-grafted anti-GM₂ antibody Lchain variable regions that showed high binding activity against GM₂,certain amino acid residues which cannot be specified by deduction fromknown human CDR-grafted antibody production examples have been replacedwith amino acids found in the mouse L chain variable region. Thus,obviously, it was very important, in human CDR-grafted anti-GM₂ antibodyproduction, to identify these amino acid residues.

Furthermore, the fact that the human CDR-grafted anti-GM₂ antibodieshaving those human CDR-grafted anti-GM₂ antibody H chain and L chainvariable regions based on the known human antibody variable regioncommon sequences showed high antigen binding activity is proof of theusefulness of the present process in human CDR-grafted antibodyproduction.

(6) Acquisition of Cell Lines for Stable Production of Human CDR-GraftedAnti-GM₂ Antibodies

Based on the results of Paragraph 4 (5) of Example 3, two cell lines,KM8966 and KM8967, capable of stably expressing KM8966, which has theamino acid sequence set forth in SEQ ID NO:60 as the H chain variableregion and the amino acid sequence set forth in SEQ ID NO:83 as the Lchain variable region, and KM8967, which has the amino acid sequence setforth in SEQ ID NO:60 as the H chain variable region and the amino acidsequence set forth in SEQ ID NO:90 as the L chain variable region,respectively as human CDR-grafted anti-GM₂ antibodies having higherantigen-binding activity than the human CDR-grafted anti-GM₂ antibodydescribed in Example 2 were obtained in the following manner.

Three μg each of the plasmids pT796HLCDRLm-28 and pT796HLCDRHSGLobtained in Paragraph 4 (5) of Example 3 were respectively added to 10μl of 20 mM Tris-hydrochloride buffer (pH 8.5) containing 100 mMpotassium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units ofthe restriction enzyme BamHI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. Each reactionmixture was subjected to ethanol precipitation, the precipitate wasadded to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 unitsof the restriction enzyme XhoI (Takara Shuzo) was further added, and thereaction was allowed to proceed at 37° C. for 1 hour. Each reactionmixture was fractionated by agarose gel electrophoresis, and about 1 μgof a BamHI-XhoI fragment about 4.93 kb in size was recovered.

The transformants KM8966 and KM8967 have been deposited with NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology as of May 23, 1995 under the deposit numbers FERMBP-5105, and FERM BP-5107, respectively.

Then, 3 μg of the plasmid pKANTEX93 obtained in Paragraph 1 of Example 3was added to 10 μl of 20 mM Tris-hydrochloride buffer (pH 8.5)containing 100 mM potassium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme BamHI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was subjected to ethanol precipitation, the precipitatewas added to 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride and 1 mMDTT, 10 units of the restriction enzyme XhoI (Takara Shuzo) was furtheradded, and the reaction was allowed to proceed at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis, andabout 1 μg of a BamHI-XhoI fragment about 8.68 kb in size was recovered.

Then, 0.1 μg of the BamHI-XhoI fragment of pT796HLCDRLm-28 orpT796HLCDRHSGL and 0.1 μg of the BamHI-XhoI fragment of pKANTEX93, eachobtained as mentioned above, were added to a total of 20 μl ofsterilized water and ligated to each other using Ready-To-Go T4 DNALigase (Pharmacia Biotech). Each recombinant plasmid DNA solution thusobtained was used to transform Escherichia coli HB101. In this way, theplasmids pKANTEX796HLCDRLm-28 and pKANTEX796HLCDRHSGL shown in FIG. 107were obtained.

Then, 4 μg each of the above plasmids pKANTEX796HLCDRLm-28 andpKANTEX796HLCDRHSGL were respectively used to transform YB2/0 (ATCC CRL1581) cells according to the procedure described in Paragraph 11 ofExample 1 and, after final selection using G418 (0.5 mg/ml) and MTX (200nM), a transformant cell line, KM8966, capable of producing about 40μg/ml of KM8966, i.e. the pKANTEX796HLCDRLm-28-derived human CDR-graftedanti-GM₂ antibody, and a transformant cell line, KM8967, capable ofproducing about 30 μg/ml of KM8967, i.e. the pKANTEX796HLCDRHSGL-derivedhuman CDR-grafted anti-GM₂ antibody, were obtained.

(7) Purification of Human CDR-Grafted Anti-GM₂ Antibodies KM8966 andKM8967

The transformant cell lines KM8966 and 8967 obtained in Paragraph 4 (6)of Example 3 were respectively suspended in GIT medium (NipponPharmaceutical) containing 0.5 mg/ml G418 and 200 nM MTX and, accordingto the procedure of Paragraph 11 of Example 1, 18 mg of purified humanCDR-grafted anti-GM₂ antibody KM8966 and 12 mg of purified KM8967 wereobtained each from about 0.5 liter of culture fluid. Three μg each ofthe purified human CDR-grafted anti-GM₂ antibodies obtained and thehuman anti-GM₂ chimera antibody KM966 were subjected to electrophoresisby the known method [Laemli, Nature, 227, 680 (1979)] for molecularweight determination. The results are shown in FIG. 108. As shown, underreducing conditions, both antibody H chains showed a molecular weight ofabout 50 kilodaltons and both antibody L chains showed a molecularweight of about 25 kilodaltons. Expression of H and L chains of correctmolecular weights was thus confirmed. Under non-reducing conditions,both human CDR-grafted anti-GM₂ antibodies showed a molecular weight ofabout 150 kilodaltons and it was thus confirmed that antibodies eachcomposed of two H chains and two L chains and having a correct size hadbeen expressed. Furthermore, the H and L chains of each humanCDR-grafted anti-GM₂ antibody were analyzed for N-terminal amino acidsequence by automatic Edman degradation using a protein sequencer(Applied Biosystems model 470A), whereby an amino acid sequencededucible from the base sequence of the variable region DNA constructedwas revealed.

5. In vitro Reactivity of Human CDR-Grafted Anti-GM₂ Antibodies KM8966and KM8967 Against GM₂

The human anti-GM₂ chimera antibody KM966 and the purified humanCDR-grafted anti-GM₂ antibodies KM8966 and KM8967 were tested forreactivity against GM₂ by ELISA as described in Paragraph 11 ofExample 1. The results are shown in FIG. 109. GM₂ (N-acetyl-Gm₂) usedwas purified from cultured cell line HPB-ALL [Oboshi et al.,Tanpakushitsu, Kakusan & Koso (Protein, Nucleic acid & Enzyme), 23, 697(1978)] in accordance with the known method [J. Biol. Chem., 263, 10915(1988)]. As shown, it was found that the purified human CDR-graftedanti-GM₂ antibody KM8966 exerted the binding activity comparable to thatof the human anti-GM₂ chimera antibody KM966. On the other hand, thebinding activity of purified human CDR-grafted anti-GM₂ antibody KM8967was about 1/4 to 1/5 of that of the human anti-GM₂ chimera antibodyKM966.

6. Reaction Specificity of Human CDR-Grafted Anti-GM₂ Antibodies KM8966and KM8967

The human anti-GM₂ chimera antibody KM966 and the human CDR-graftedanti-GM₂ antibodies KM8966 and KM8967 were tested for reactivity againstthe gangliosides GM₁, N-acetyl-GM₂, N-glycolyl-GM₂, N-acetyl-GM₃,N-glycolyl-GM₃, GD_(1a), GD_(1b) (Iatron), GD₂, GD₃ (Iatron) and GO_(1b)(Iatron) by ELISA as described in Paragraph 11 of Example 1. The resultsare shown in FIG. 110. GM₁ and GD_(1a) were purified from bovine brain,N-acetyl-GM₂ from cultured cell line HPB-ALL [Oboshi et al.,Tanpakushitsu, Kakusan & Koso (Protein, Nucleic acid & Enzyme), 23, 697(1978)], N-glycolyl-GM₂ and N-glycolyl-GM₃ from mouse liver,N-acetyl-GM₃ canine erythrocytes, and GD₂ from cultured cell line IMR32(ATCC CCL127), respectively by the per se known method [J. Biol. Chem.,263, 10915 (1988)]. Each antibody was used in a concentration of 10μg/ml.

As shown in FIG. 110, it was confirmed that the human CDR-graftedanti-GM₂ antibodies KM8966 and KM8967 react specifically with GM₂(N-acetyl-GM₂ and N-glycolyl-GM₂) like the human anti-GM₂ chimeraantibody KM966.

7. Reactivity of Human CDR-Grafted Anti-GM₂ Antibodies KM8966 and KM8967Against Cancer Cells

The human lung small cell carcinoma culture cell line SBC-3 (JCRB 0818)(1×10⁶ cells) was suspended in PBS, the suspension was placed in amicrotube (TREF) and centrifuged (1200 rpm, 2 minutes). To thethus-washed cells was added 50 μl (50 μg/ml) of the human anti-GM₂chimera antibody KM966 or the purified human CDR-grafted anti-GM₂antibody KM8966 or KM8967, followed by stirring and 1 hour of standingat 4° C. After the above reaction step, the cells were washed threetimes with PBS, each time followed by centrifugation. Then, 20 μl offluorescein isocyanate-labeled protein A (30-fold dilution, BoehringerMannheim) was added and, after stirring, the reaction was allowed toproceed at 4° C. for 1 hour. Thereafter, the cells were washed threetimes with PBS, each time followed by centrifugation, then furthersuspended in PBS and subjected to analysis using a flow cytometer, EPICSElite (Coulter). In a control run, the above procedure was followedwithout addition of the human CDR-grafted anti-GM₂ antibody andanalyzed. The results are shown in FIG. 111. It was found that thepurified human CDR-grafted anti-GM₂ antibodies KM8966 and KM8967strongly reacted with the human lung small cell carcinoma culture cellline SBC-3 like the human anti-GM₂ chimera antibody KM966.

8. In vitro Antitumor Activity of Human CDR-Grafted Anti-GM₂ AntibodiesKM8966 and KM8967: Complement Dependent Cytotoxicity (CDC)

(1) Preparation of Target Cells

The target cells SBC-3, cultured in RPMI1640 medium supplemented with10% FCS, were adjusted to a cell concentration of 5×10⁶ cells/500 μl,3.7 MBq of ⁵¹ Na₂ CrO₄ (Daiichi Pure Chemicals Co., Ltd.) was addedthereto. Then, the reaction was allowed to proceed at 37° C. for 1 hour,and the cells were washed three times with the medium. The cells werethen allowed to stand in the medium at 4° C. for 30 minutes and, aftercentrifugation, the medium was added to adjust the cell concentration to1×10⁶ cells/ml.

(2) Preparation of the Complement

Sera from healthy subjects were combined and used as a complementsource.

(3) CDC Activity Measurement

The chimeric human anti-GM₂ antibody KM966 or purified human CDR-graftedanti-GM₂ antibody KM8966 or KM8967 was added to wells of 96-wellU-bottom plates within the final concentration range of 0.05 to 50 μg/mland then 50 μl (5×10⁴ cells/well) of the target cells prepared in (1)were added to each well. The reaction was allowed to proceed at roomtemperature for 1 hour. After centrifugation, the supernatants werediscarded, the human complement obtained in (2) was added to each wellto give a final concentration of 15% v/v, and the reaction was allowedto proceed at 37° C. for 1 hour. After centrifugation, the amount of ⁵¹Cr in each supernatant was determined using a gamma counter. The amountof spontaneously dissociated ⁵¹ Cr was determined by adding to thetarget cells the medium alone in stead of the antibody and complementsolutions and measuring the amount of ⁵¹ Cr in the supernatant in thesame manner as mentioned above. The total amount of dissociated ⁵¹ Crwas determined by adding to the target cells 1 N hydrochloric acid instead of the antibody and complement solutions and measuring the amountof ⁵¹ Cr in the supernatant in the same manner as mentioned above. TheCDC activity was calculated as follows: ##EQU3##

The results thus obtained are shown in FIG. 112. It was shown that CDCactivity of the human CDR-grafted anti-GM₂ antibodies KM8966 and KM8967was lower than that of the chimeric human anti-GM₂ antibody KM966.

9. In vitro Antitumor Activity of Human CDR-Grafted Anti-GM₂ AntibodiesKM8966 and KM8967: Antibody Dependent Cell Mediated Cytotoxicity (ADCC)

(1) Preparation of Target Cells

The target cells SBC-3 cultured in RPMI1640 medium supplemented with 10%FCS were adjusted to a cell concentration of 1×10⁶ cells/500 μl, 3.7 MBqof ⁵¹ Na₂ CrO₄ (Daiichi Pure Chemicals Co., Ltd.) was added thereto.Then, the reaction was allowed to proceed at 37° C. for 1 hour and thecells were washed three times with the medium. The cells were thenallowed to stand in the medium at 4° C. for 30 minutes and then, aftercentrifugation, the medium was added to adjust the cell concentration to2×10⁵ cells/ml.

(2) Preparation of Effector Cells

Human venous blood (50 ml) was collected, 0.5 ml of heparin sodium(Takeda Chemical Industries; 1,000 units/ml) was added, and the mixturewas gently stirred. This mixture was overlaid on Polymorphprep (Nycomed)and centrifuged to separate the lymphocyte layer (PBMC). The resultinglymphocytes were washed three times by centrifugation with RPMI1640medium supplemented with 10% FCS, and the cells were suspended in themedium (5×10⁶ cells/ml) for use as effector cells.

(3) ADCC Activity Measurement

The chimeric human anti-GM₂ antibody KM966 or purified human CDR-graftedanti-GM₂ antibodies KM8966 or KM8967 was added to wells of 96-wellU-bottom plates within the final concentration range of 0.05 to 50 μg/mland then 50 μl (1×10⁴ cells/well) of the target cell suspension preparedin (1) and 100 μl (5×10⁵ cells/well) of the effector cell suspensionprepared in (2) were added to each well. The reaction was allowed toproceed at 37° C. for 4 hours and, after centrifugation, the amount of⁵¹ Cr in each supernatant was measured using a gamma counter. The amountof spontaneously dissociated ⁵¹ Cr was determined by adding to thetarget cells the medium alone in lieu of the antibody and effector cellsand measuring the amount of ⁵¹ Cr in the supernatant in the same manneras mentioned above. The total amount of dissociated ⁵¹ Cr was determinedby adding to the target cells 1 N hydrochloric acid in lieu of theantibody and effector cells and measuring the amount of ⁵¹ Cr in thesupernatant in the same manner as mentioned above. The ADCC activity wascalculated as follows: ##EQU4##

The results thus obtained are shown in FIG. 113. The human CDR-graftedanti-GM₂ antibody KM8966 showed ADCC activity comparable to that of thechimeric human anti-GM₂ antibody KM966, whereas the human CDR-graftedanti-GM₂ antibody KM8967 showed ADCC activity slightly lower than thatof the chimeric human anti-GM₂ antibody KM966.

EXAMPLE 4 Production of Human CDR-Grafted Anti-GM₂ Antibodies

The human CDR-grafted anti-GM₂ antibodies KM8966 and KM8967 showedantigen binding activity (ELISA), binding specificity and ADCC activitycomparable to those of the chimeric human anti-GM₂ antibody KM966, whileits CDC activity was lower than that of the chimeric antibody. In orderto improve the CDC activity, human CDR-grafted anti-GM₂ antibodies wereproduced in the following manner.

1. Modification of Human CDR-Grafted Anti-GM₂ Antibody KM8966 H ChainVariable Region

Among the human CDR-grafted anti-GM₂ antibodies prepared in Example 3,the antibody KM8966 showing higher CDC activity was subjected to aminoacid mutation at the H chain variable region (SEQ ID NO:60) in order toimprove CDC activity. The amino acids to be mutated were selected atrandom with reference to the results of various mutation obtained inExample 3 and a computer model for the variable region of mouse KM796.Mutations were introduced by PCR method using as a template 1 ng of theplasmid pBSH10 containing the human CDR-grafted anti-GM₂ antibody Hchain variable region obtained in Paragraph 4 (2) of Example 3 and usingas a primer antisense and sense synthetic DNA containing mutationsdescribed in Paragraph 4 (3) of Example 3.

The reaction was carried out in the same manner as described inParagraph 4 (3) of Example 3 using the synthetic DNA of SEQ ID NO:91 asthe mutant antisense primer and the synthetic DNA of SEQ ID NO:92 as themutant sense primer to obtain the plasmid pBSHM1 containing version HM1,shown in SEQ ID NO:93, of the human CDR-grafted anti-GM₂ antibody Hchain variable region. In the amino acid sequence of the version HM1,arginine in position 38, alanine in position 40, glutamine in position43 and glycine in position 44 in the FR shown in SEQ ID NO:60 werereplaced with lysine, serine, lysine and serine, respectively, that arefound in the mouse KM796 H chain variable region. The plasmid pBSHM2containing version HM2, shown in SEQ ID NO:96, of the human CDR-graftedanti-GM₂ antibody H chain variable region was obtained following thereaction described in Paragraph 4 (3) of Example 3 using the syntheticDNA of SEQ ID NO:94 as the mutant antisense primer and the synthetic DNAof SEQ ID NO:95 as the mutant sense primer. In the amino acid sequenceof the version HM2, arginine in position 38 and alanine in position 40in the FR shown in SEQ ID NO:60 were replaced with lysine and serine,respectively, that are found in the mouse KM796 H chain variable region.

The plasmid pBSHM3 containing version HM3, shown in SEQ ID NO:99, of thehuman CDR-grafted anti-GM₂ antibody H chain variable region was obtainedfollowing the reaction described in Paragraph 4 (3) of Example 3 usingthe synthetic DNA of SEQ ID NO:97 as the mutant antisense primer and thesynthetic DNA of SEQ ID NO:98 as the mutant sense primer. In the aminoacid sequence of the version HM3, valine in position 68 and isoleucinein position 70 in the FR shown in SEQ ID NO:60 were replaced withalanine and leucine, respectively, that are found in the mouse KM796 Hchain variable region.

The plasmid pBSHM31 containing version HM31, shown in SEQ ID NO:100, ofthe human CDR-grafted anti-GM₂ antibody H chain variable region wasobtained following the reaction described in Paragraph 4 (3) of Example3 using 1 ng of the plasmid pBSHM3 as the template, the synthetic DNA ofSEQ ID NO:91 as the mutant antisense primer and the synthetic DNA of SEQID NO:92 as the mutant sense primer. In the amino acid sequence of theversion HM31, arginine in position 38, alanine in position 40, μlutaminein position 43 and glycine in position 44 in the FR of the version HM3were replaced with lysine, serine, lysine and serine, respectively, thatare found in the mouse KM796 H chain variable region.

Further, the plasmid pBSHM32 containing version HM32, shown in SEQ IDNO:101, of the human CDR-grafted anti-GM₂ antibody H chain variableregion was obtained following the reaction described in Paragraph 4 (3)of Example 3 using 1 ng of the plasmid pBSHM3 as the template, thesynthetic DNA of SEQ ID NO:94 as the mutant antisense primer and thesynthetic DNA of SEQ ID NO:95 as the mutant sense primer. In the aminoacid sequence of the version HM32, arginine in position 38 and alaninein position 40 in the FR of the version HM3 were replaced with lysineand serine, respectively, that are found in the mouse KM796 H chainvariable region.

2. Evaluation of CDC Activity of Various Human CDR-Grafted Anti-GM₂Antibodies Having Mutations in the Human CDR-Grafted Anti-GM₂ Antibody HChain Variable Region

(1) Construction of Expression Vectors

Expression vectors for various human CDR-grafted anti-GM₂ antibodiescontaining the H chain variable region of human CDR-grafted anti-GM₂antibodies having various mutations obtained in Paragraph 1 of Example 4and the L chain variable region of IM8966 (SEQ ID NO:83) were preparedin the following manner.

3 μg each of the plasmids pBSHM1, pBSHM2, pBSHM3, pBSHM31 and pBSHM32obtained in Paragraph 1 of Example 4 were dissolved in 10 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of ApaI (Takara Shuzo) were added thereto and themixture was allowed to react at 37° C. for 1 hour. The resulting mixturewas subjected to ethanol precipitation and the thus-obtained precipitatewas dissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT,100 μg/ml BSA and 0.01% of Triton X-100. 10 units of NotI (Takara Shuzo)were further added thereto to allow the mixture to react at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis to recover about 0.2 μg of the ApaI-NotI fragment ofabout 0.44 kb.

Then, 3 μg of the plasmid pKANTEX796HLCDRLm-28 obtained in Paragraph 4(5) of Example 3 was dissolved in 10 μl of 10 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10units of ApaI (Takara Shuzo) were added thereto and the mixture wasallowed to react at 37° C. for 1 hour. The resulting mixture wassubjected to ethanol precipitation and the thus-obtained precipitate wasdissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT,100 μg/ml BSA and 0.01% of Triton X-100. 10 units of NotI (Takara Shuzo)were added thereto to allow the mixture to react at 37° C. for 1 hour.The reaction mixture was fractionated by agarose gel electrophoresis torecover about 1 μg of the ApaI-NotI fragment of about 13.14 kb.

About 0.1 μg each of the thus-obtained ApaI-NotI fragment of pBSHM1,pBSHM2, pBSHM3, pBSHM31 and pBSHM32 and 0.1 μg of the ApaI-NotI fragmentof pKANTEX796HLCDRLm-28 were added in a total of 20 μl of sterilizedwater and ligated to each other using Ready-To-Go T4 DNA Ligase(Pharmacia Biotech). Each of the resulting recombinant plasmid DNAsolutions was used to transform Escherichia coli HB101 and plasmids,pKANTEX796HM1Lm-28, pKANTEX796HM2Lm-28, pKANTEX796HM3Lm-28,pKANTEX796HM31Lm-28 and pKANTEX796HM32Lm-28 shown in FIG. 114 wereobtained.

(2) Expression of Mutant Versions of Human CDR-Grafted Anti-GM₂Antibodies

4 μg each of the plasmids pKANTEX796HM1Lm-28, pKANTEX796HM2Lm-28,pKANTEX796HM3Lm-28, pKANTEX796HM31Lm-28 and pKANTEX796HM32Lm-28 obtainedin Paragraph 2 (1) of Example 4 were used to transform YB2/0 cells (ATCCCRL 1581) in accordance with the method as described in Paragraph 11 ofExample 1. The cells were ultimately selected using G418 (0.5 mg/ml) andMTX (200 nM) to obtain about 2 to 5 μg/ml of transformants capable ofproducing human CDR-grafted anti-GM₂ antibodies derived from thecorresponding expression vectors.

(3) Purification of Mutant Versions of Human CDR-Grafted Anti-GM₂Antibodies

Cells of each transformant obtained in Paragraph 2 (2) of Example 4 weresuspended in GIT medium (Nihon Pharmaceutical) containing 0.5 mg/ml G418and 200 nM MTX and about 1 to 3 mg of purified human CDR-graftedanti-GM₂ antibodies were obtained from about 0.6 liter of the culturebroth in accordance with the method described in Paragraph 11 ofExample 1. The human CDR-grafted anti-GM₂ antibodies derived from theplasmids pKANTEX796HM1Lm-28, pKANTEX796HM2Lm-28, pKANTEX796HM3Lm-28,pKANTEX796HM31Lm-28 and pKANTEX796HM32Lm-28 are hereinafter referred toas M1-28, M2-28, M3-28, M31-28 and M32-28, respectively. 4 μg each ofthe purified human CDR-grafted anti-GM₂ antibodies, the humanCDR-grafted anti-GM₂ antibody KM8966 and the chimeric human anti-GM₂antibody KM966 were electrophoresed by the conventional method [Laemmli:Nature, 227, 680 (1970)] for molecular weight checking. The results areshown in FIG. 115. As shown in FIG. 115, under reducing conditions, themolecular weight of the antibody H chain was about 50 KDa and themolecular weight of the antibody L chain was about 25 KDa, thusconfirming the expression of the H chain and L chain having the correctmolecular weight. Under nonreducing conditions, the molecular weight ofthe human CDR-grafted anti-GM₂ antibodies was about 150 KDa, confirmingthat the antibody expressed was composed of two H chains and two Lchains and was correct in size. The N-terminal amino acid sequence ofthe H and L chains of each purified human CDR-grafted anti-GM₂antibodies was examined by automatic Edman degradation using a proteinsequencer (Applied Biosystems model 470A). As a result, it was confirmedthat the amino acid sequence was consistent with that deduced from thesynthesized variable region DNA sequence.

(4) CDC Activity of Mutant Versions of Human CDR-Grafted Anti-GM₂Antibodies

CDC activity of the mutant versions of the human CDR-grafted anti-GM₂antibodies obtained in Paragraph 2 (3) of Example 4, the humanCDR-grafted anti-GM₂ antibody KM8966 and the chimeric human anti-GM₂antibody KM966 was measured in accordance with the method described inParagraph 8 of Example 3. The results are shown in FIG. 116. As shown inFIG. 116, it was found that, among the mutant versions of the humanCDR-grafted anti-GM₂ antibodies, the human CDR-grafted anti-₂ GMantibody M2-28 derived from the plasmid pKANTEX796HM2Lm-28 showed thehighest CDC activity which was higher than that of the human CDR-graftedanti-GM₂ antibody KM8966 prepared in Example 3. This result indicatesthat the mutated amino acid residues in positions 38 and 40 in the FR ofthe version HM2 among the various mutant versions prepared in Paragraph1 of Example 4 play an important role for improving CDC activity. It wasassumed from the computer model for the variable region of mouse KM796that the mutation of the amino acid residues in positions 38 and 40 inthe FR of the version HM2 would influence on the entire structure of thevariable region since these amino acid residues are located at the sitewhich interacts with the L chain variable region. Recent study of theproduction of human CDR-grafted antibody reveals that the amino acidresidues which affect the structure of the antibody varies in eachantibody. No method for precisely predicting such amino acid residueshas been established and the above results provide a significant findingfor the production of the human CDR-grafted antibody.

The human CDR-grafted anti-GM₂ antibody M2-28 derived from the plasmidpKANTEX796HM2Lm-28 was designated as KM8970 and the antibodyKM8970-producing transformant KM8970 has been deposited with NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology as of under the deposit number FERM BP-.

3. Modification of Human CDR-Grafted Anti-GM₂ Antibody KM8966 L ChainVariable Region

The human CDR-grafted anti-GM₂ antibody KM8966 prepared in Example 3 wassubjected to amino acid mutation in the L chain variable region (SEQ IDNO:83) to improve CDC activity. As an amino acid to be mutated, serineresidue in position 59 was selected based on the results of variousmutations obtained in Example 3 which suggested that it was important tosupport the structure of CDR2 for the human CDR-grafted anti-GM₂antibody activity. Mutations were introduced by PCR method using as atemplate 1 ng of the plasmid pBSLm-28 containing the human CDR-graftedanti-GM₂ antibody L chain variable region obtained in Paragraph 4 (3) ofExample 3 and using as a primer antisense and sense synthetic DNAcontaining mutations described in Paragraph 4 (3) of Example 3.

The reaction was carried out in the same manner as described inParagraph 4 (3) of Example 3 using the synthetic DNA of SEQ ID NO:102 asthe mutant antisense primer and the synthetic DNA of SEQ ID NO:103 asthe mutant sense primer to obtain the plasmid pBSLm-28 No.1, containingversion Lm-28 No.1, shown in SEQ ID NO:104, of the human CDR-graftedanti-GM₂ antibody L chain variable region. In the amino acid sequence ofthe version Lm-28 No.1, serine in position 59 in the FR shown in SEQ IDNO:83 was replaced with alanine that is found in the mouse KM796 L chainvariable region.

4. Evaluation of CDC Activity of Human CDR-Grafted Anti-GM₂ AntibodyHaving New Mutation in Human CDR-Grafted Anti-GM₂ Antibody L ChainVariable Region

(1) Construction of Expression Vectors

Expression vectors for the human CDR-grafted anti-GM₂ antibodycontaining the human CDR-grafted anti-GM₂ antibody L chain variableregion having the mutation obtained in Paragraph 3 of Example 4 and thehuman CDR-grafted anti-GM₂ antibody H chain variable region wereobtained in the following manner. 6 μg of the plasmid pBSLm-28 No.1obtained in Paragraph 3 of Example 4 was dissolved in 10 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 100 mM sodium chloride, 10mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA. 10 units each ofEcoRI (Takara Shuzo) and SplI (Takara Shuzo) were added thereto to allowthe mixture to react at 37° C. for 1 hour. The reaction mixture wasfractionated by agarose gel electrophoresis to recover about 0.4 μg ofthe EcoRI-SplI fragment of about 0.39 kb.

Then, 3 μg each of the plasmid pKANTEX796HLCDRLm-28 obtained inParagraph 4 (5) of Example 3 and the plasmids pKANTEX796HM1Lm-28,pKANTEX796HM2Lm-28 and pKANTEX796HM3Lm-28 obtained in Paragraph 2 (1) ofExample 4 were dissolved in 10 μl of 50 mM Tris-hydrochloride buffer (pH7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride and 1mM DTT and 100 μg/ml BSA, 10 units each of EcoRI (Takara Shuzo) and SplIwere added thereto and the mixture was allowed to react at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis to recover about 1 μg of the EcoRI-SplI fragment ofabout 13.19 kb.

A 0.1 μg portion each of the thus-obtained EcoRI-SplI fragment ofpBSLm-28 No.1 and 0.1 μg of the EcoRI-SplI of pKANTEX796HLCDRLm-28,pKANTEX796HM1Lm-28, pKANTEX796HM2Lm-28 and pKANTEX796HM3Lm-28 were addedin a total of 20 μl of sterilized water and ligated to each other usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). Each of the resultingrecombinant plasmid DNA solutions was used to transform Escherichia coliHB101 and the plasmids pKANTEX796HLm-28 No.1, pKANTEX796HM1Lm-28 No.1,pKANTEX796HM2Lm-28 No.1 and pKANTEX796HM3Lm-28 No.1 shown in FIG. 117were obtained.

(2) Expression of Human CDR-Grafted Anti-GM₂ Antibodies Having Mutationsin the L Chain Variable Region

4 μg each of the plasmids pKANTEX796HLm-28 No.1, pKANTEX796HM1Lm-28No.1, pKANTEX796HM2Lm-28 No.1 and pKANTEX796HM3Lm-28 No.1 obtained inParagraph 4 (1) of Example 4 was used to transform YB2/0 cells (ATCC CRL1581) in accordance with the method as described in Paragraph 11 ofExample 1. The cells were ultimately selected using G418 (0.5 mg/ml) andMTX (200 nM) to obtain about 2 to 5 μg/ml of transformants capable ofproducing human CDR-grafted anti-GM₂ antibodies derived from thecorresponding expression vectors.

(3) Purification of Human CDR-Grafted Anti-GM₂ Antibodies HavingMutation in the L Chain Variable Region

Cells of each transformant obtained in Paragraph 4 (2) of Example 4 weresuspended in GIT medium (Nihon Pharmaceutical) containing 0.5 mg/ml G418and 200 nM MTX and about 1 to 3 mg of purified human CDR-graftedanti-GM₂ antibodies were obtained from about 0.6 liter of the culturebroth in accordance with the method described in Paragraph 11 ofExample 1. The human CDR-grafted anti-GM₂ antibodies derived from theplasmids pKANTEX796HLm-28 No.1, pKANTEX796HM1Lm-28 No.1,pKANTEX796HM2Lm-28 No.1 and pKANTEX796HM3Lm-28 No.1 are hereinafterreferred to as h796H-No.1, M1-No.1, M2-No.1 and M3-No.1, respectively. 4μg each of the purified human CDR-grafted anti-GM₂ antibodies and thechimeric human anti-GM₂ antibody KM966 was electrophoresed by theconventional method [Laemmli: Nature, 227, 680 (1970)] for molecularweight checking. The results are shown in FIG. 118. As shown in FIG.118, under reducing conditions, the molecular weight of the antibody Hchain was about 50 KDa and the molecular weight of the antibody L chainwas about 25 KDa, thus confirming the expression of the H chain and Lchain having the correct molecular weight. Under nonreducing conditions,the molecular weight of the human CDR-grafted anti-GM₂ antibodies wasabout 150 KDa, confirming that the antibody expressed was composed oftwo H chains and two L chains and was correct in size. The N-terminalamino acid sequence of the H and L chains of each purified humanCDR-grafted anti-GM₂ antibodies was examined by automatic Edmandegradation using a protein sequencer (Applied Biosystems model 470A).As a result, it was confirmed that the amino acid sequence wasconsistent with that deduced from the synthesized variable region DNAsequence.

(4) CDC Activity of Human CDR-Grafted Anti-GM₂ Antibodies HavingMutation in the L Chain Variable Region

CDC activity of the human CDR-grafted anti-GM₂ antibodies havingmutation in the L chain variable region obtained in Paragraph 4 (3) ofExample 4, the human CDR-grafted anti-GM₂ antibody KM8970 obtained inParagraph 2 of Example 4, the human CDR-grafted anti-GM₂ antibody KM8966and the chimeric human anti-GM₂ antibody KM966 was measured inaccordance with the method described in Paragraph 8 of Example 3. Theresults are shown in FIG. 119. Comparing CDC activity of KM8966 withthat of h796H-No.1, it was found that h796H-No.1 in which mutation wasintroduced into only the L chain variable region showed improved CDCactivity. Among the mutant antibodies having mutations in both of the Lchain variable region and the H chain variable region, M2-No.1 havingmutation in the human CDR-grafted anti-GM₂ antibody KM8970 H and L chainvariable region showed the highest CDC activity, which was comparable toor higher than that of KM8970. These results indicates that the mutatedamino acid residue in position 59 in the FR of the L chain variableregion prepared in Paragraph 3 of Example 4 played an important role forimproving its CDC activity and it interacted with the mutated amino acidresidue in the H chain variable region of KM8970 for improving its CDCactivity. It was not assumed from the computer model for the variableregion of mouse KM796 that the mutation of the amino acid residue inposition 59 in the FR of the version Lm-28 No.1 would be involved indirect action with antigen GM₂ and interaction with each CDR residue.However, the above results suggested that the amino acid residue atposition 59 in the FR was quite important for maintaining the entirestructure of the variable region. There has been no report on theproduction of a humanized antibody which shows improved biologicalactivity by modifying the above-described amino acid residue in the Lchain variable region. Further, since it is impossible to predict intheory the amino acid residue to be mutated using, for example, thecomputer model, the above findings will provide an important indicationfor the production of human CDR-grafted antibody.

The human CDR-grafted anti-GM₂ antibody M2-No.1 derived from the plasmidpKANTEX796HM2Lm-28 No.1 was designated as KM8969 and the antibodyKM8969-producing transformant KM8969 has been deposited with NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology as of under the deposit number FERM BP-.

5. In vitro Reactivity of Human CDR-Grafted Anti-GM₂ Antibodies KM8969and KM8970 with GM₂

Reactivities of the chimeric human anti-GM₂ antibody KM966 and the humanCDR-grafted anti-GM₂ antibodies KM8969 and KM8970 with GM₂ were measuredin accordance with the method described in Paragraph 5 of Example 3. Theresults are shown in FIG. 120. As shown in FIG. 120, the humanCDR-grafted anti-GM₂ antibodies KM8969 and KM8970 showed bindingactivity comparable to that of the chimeric human anti-GM₂ antibodyKM966.

6. Reaction specificity of human CDR-grafted anti-GM₂ antibodies KM8969and KM8970

The chimeric human anti-GM₂ antibody KM966 and the human CDR-graftedanti-GM₂ antibodies KM8969 and KM8970 were examined for reactivity withvarious gangliosides in accordance with the method described inParagraph 6 of Example 3. The results are shown in FIG. 121. As shown inFIG. 121, it was found that the human CDR-grafted anti-GM₂ antibodiesKM8969 and KM8970 specifically reacted with GM₂ (N-acetyl GM₂ andN-glycolyl GM₂) like the chimeric human anti-GM₂ antibody KM966.

7. Reactivity of Human CDR-grafted Anti-GM₂ Antibodies KM8969 and KM8970with Cancer Cells

The chimeric human anti-GM₂ antibody KM966 and the human CDR-graftedanti-GM₂ antibodies KM8969 and KM8970 were examined for reactivity withthe human lung small cell carcinoma cell line SBC-3 (JCRB 0818) usingfluorescein isocyanate-labeled rabbit anti-human IgG antibody (Dako) asa second antibody in accordance with the method described in Paragraph 7of Example 3. The results are shown in FIG. 122. As shown in FIG. 122,the human CDR-grafted anti-GM₂ antibodies KM8969 and KM8970 stronglyreacted with the human lung small cell carcinoma cell line SBC-3 likethe chimeric human anti-GM₂ antibody KM966.

8. In Vitro Antitumor Effect of Human CDR-grafted Anti-GM₂ AntibodiesKM8969 and KM8970: Antibody Dependent Cell Mediated Cytotoxicity (ADCC)

The chimeric human anti-GM₂ antibody KM966 and the human CDR-graftedanti-GM₂ antibodies KM8966, KM8969 and KM8970 were examined for ADCCactivity against the human lung small cell carcinoma cell line SBC-3(JCRB 0818) in accordance with the method described in Paragraph 9 ofExample 3. The results are shown in FIG. 123. As shown in FIG. 123, thehuman CDR-grafted anti-GM₂ antibodies KM8969 and KM8970 showed ADCCactivity comparable to that of the chimeric human anti-GM₂ antibodyKM966.

9. In Vitro Antitumor Effect of Humanized Anti-GM₂ Antibodies:Comparison of CDC Activities

The CDC activities of the humanized anti-GM₂ antibodies KM966, KM8966,KM8969 and KM8970 prepared in Examples 3 and 4 were measured withprolonging the reaction period. Namely, after the human complement wasadded, the reaction was carried out for 4 hours in accordance with themethod described in Paragraph 8 of Example 3. The results are shown inFIG. 124. As shown in FIG. 124, the human CDR-grafted anti-GM₂antibodies KM8966, KM8969 and KM8970 showed the CDC activity comparableto that of the chimeric human anti-GM₂ antibody KM966 when the antibodyconcentration was not less that 5 μg/ml. These results suggest that thehuman anti-GM₂ antibodies prepared in Examples are useful for humancancer treatment.

As described above, the production of humanized anti-GM₂ antibodies andthe results of their activities as described in Examples show that thethus established humanized anti-GM₂ antibodies are useful for humancancer treatment.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 104                                           - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 449 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 19..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - #BY SIMILARITY WITH KNOWN SEQUENCE     #AN ESTABLISHED CONSENSUS                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 31..35                                                          (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 1"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 50..66                                                          (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 2"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 99..109                                                         (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 3"                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - CTCCACAGTC CCTGAAGACA CTGACTCTAA CC ATG GGA TGG AGC - # TGG ATC TTT           53                                                                          #Met Gly Trp Ser Trp Ile Phe                                                  15                                                                            - CTC TTC CTC CTG TCA GGA ACT GCA GGT GTC CT - #C TCT GAG GTC CAG CTG          101                                                                          Leu Phe Leu Leu Ser Gly Thr Ala Gly Val Le - #u Ser Glu Val Gln Leu           #         1                                                                   - CAG CAG TCT GGA CCT GAG CTG GTG AAG CCT GG - #G GCT TCA GTG AAG ATA          149                                                                          Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Ile           #  20                                                                         - TCC TGC AAG GCT TCT GGA TAC ACA TTC ACT GA - #C TAC AAC ATG GAC TGG          197                                                                          Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr As - #p Tyr Asn Met Asp Trp           #                 35                                                          - GTG AAG CAG AGC CAT GGA AAG AGC CTT GAG TG - #G ATT GGA TAT ATT TAT          245                                                                          Val Lys Gln Ser His Gly Lys Ser Leu Glu Tr - #p Ile Gly Tyr Ile Tyr           #             50                                                              - CCT AAC AAT GGT GGT ACT GGC TAC AAC CAG AA - #G TTC AAG AGC AAG GCC          293                                                                          Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Ly - #s Phe Lys Ser Lys Ala           #         65                                                                  - ACA TTG ACT GTA GAC AAG TCC TCC AGC ACA GC - #C TAC ATG GAG CTC CAC          341                                                                          Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Al - #a Tyr Met Glu Leu His           #     80                                                                      - AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TA - #C TGT GCA ACC TAC GGT          389                                                                          Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ty - #r Cys Ala Thr Tyr Gly           #100                                                                          - CAT TAC TAC GGC TAC ATG TTT GCT TAC TGG GG - #C CAA GGG ACT CTG GTC          437                                                                          His Tyr Tyr Gly Tyr Met Phe Ala Tyr Trp Gl - #y Gln Gly Thr Leu Val           #               115                                                           #      449                                                                    Thr Val Ser Ala                                                                           120                                                               - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 393 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - #BY SIMILARITY WITH KNOWN SEQUENCE     TO                                                                            #ESTABLISHED CONSENSUS                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 1"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 2"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 3"                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #TTC CTG CTA ATC AGT        48AG ATT TTC AGC                                         Met His Phe Gln Val Gln Il - #e Phe Ser Phe Leu Leu Ile Ser            10                                                                            - GCC TCA GTC ATA ATG TCC AGA GGA CAA ATT GT - #T CTC ACC CAG TCT CCA           96                                                                          Ala Ser Val Ile Met Ser Arg Gly Gln Ile Va - #l Leu Thr Gln Ser Pro           #           5  1                                                              - GCA ATC ATG TCT GCA TCT CCA GGG GAG AAG GT - #C ACC ATA ACC TGC AGT          144                                                                          Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Va - #l Thr Ile Thr Cys Ser           #     20                                                                      - GCC AGC TCA AGT GTA AGT TAC ATG CAC TGG TT - #C CAG CAG AAG CCA GGC          192                                                                          Ala Ser Ser Ser Val Ser Tyr Met His Trp Ph - #e Gln Gln Lys Pro Gly           # 40                                                                          - ACT TCT CCC AAA CTC TGG ATT TAT AGC ACA TC - #C AAC CTG GCT TCT GGA          240                                                                          Thr Ser Pro Lys Leu Trp Ile Tyr Ser Thr Se - #r Asn Leu Ala Ser Gly           #                 55                                                          - GTC CCT GCT CGC TTC AGT GGC AGT GGA TCT GG - #G ACC TCT TAC TCT CTC          288                                                                          Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gl - #y Thr Ser Tyr Ser Leu           #             70                                                              - ACA ATC AGC CGA ATG GAG GCT GAA GAT GCT GC - #C ACT TAT TAC TGC CAG          336                                                                          Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Al - #a Thr Tyr Tyr Cys Gln           #         85                                                                  - CAA AGG AGT AGT TAC CCG TAC ACG TTC GGA GG - #G GGG ACC AAG CTG GAA          384                                                                          Gln Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Gl - #y Gly Thr Lys Leu Glu           #    100                                                                      #        393                                                                  Ile Lys Arg                                                                   105                                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 443 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 19..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - #BY SIMILARITY WITH KNOWN SEQUENCE     OR                                                                            #ESTABLISHED CONSENSUS                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 31..35                                                          (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 1"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 55..66                                                          (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 2"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 99..107                                                         (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISEHD                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 3"                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - CTCCACAGTC CCTGAAGACA CTGACTCTAA CC ATG GGA TGG AGC - # TGG ATC TTT           53                                                                          #Met Gly Trp Ser Trp Ile Phe                                                  15                                                                            - CTC TTC CTC CTG TCA GGA ACT GCA GGT GTC CT - #C TCT GAG GTC CAG CTG          101                                                                          Leu Phe Leu Leu Ser Gly Thr Ala Gly Val Le - #u Ser Glu Val Gln Leu           #         1                                                                   - CAG CAG TCT GGA CCT GAG CTG GTG AAG CCT GG - #G GCT TCA GTG AAG ATA          149                                                                          Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Ile           #  20                                                                         - TCC TGC AAG GCT TCT GGA TAC ACA TTC ACT GA - #C TAC AAC ATG GAC TGG          197                                                                          Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr As - #p Tyr Asn Met Asp Trp           #                 35                                                          - GTG AAG CAG AGC CAT GGA AAG AGC CTT GAG TG - #G ATT GGA TAT ATT TAT          245                                                                          Val Lys Gln Ser His Gly Lys Ser Leu Glu Tr - #p Ile Gly Tyr Ile Tyr           #             50                                                              - CCT AAC AAT GGT GGT ACT GGC TAC AAC CAG AA - #G TTC AAG AGC AAG GCC          293                                                                          Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Ly - #s Phe Lys Ser Lys Ala           #         65                                                                  - ACA TTG ACT GTA GAC AAG TCC TCC AGC ACA GC - #C TAC ATG GAG CTC CAC          341                                                                          Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Al - #a Tyr Met Glu Leu His           #     80                                                                      - AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TA - #C TGT GCA AGA GCG GGG          389                                                                          Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ty - #r Cys Ala Arg Ala Gly           #100                                                                          - AGG TAT TAC TAC GCC TGG GAC TGG GGC CAA GG - #G ACT CTG GTC ACT GTC          437                                                                          Arg Tyr Tyr Tyr Ala Trp Asp Trp Gly Gln Gl - #y Thr Leu Val Thr Val           #               115                                                           #          443                                                                Ser Ala                                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 405 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 10..66                                                          (C) IDENTIFICATION METHOD:                                                         BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN                 #CONSENSUS     ESTABLISHED                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 157..171                                                        (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 1"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 214..261                                                        (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 2"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 358..369                                                        (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 3"                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - CATCACAGC ATG GCT GTC CTG GTG CTG TTG CTC TGC - # CTG GTG ACA TTT             48                                                                          #Leu Leu Leu Cys Leu Val Thr Phe                                              10                                                                            - CCA AGC TGT GTC CTG TCC CAA GTG CAG CTG AA - #G GAG TCA GGA CCT GGT           96                                                                          Pro Ser Cys Val Leu Ser Gln Val Gln Leu Ly - #s Glu Ser Gly Pro Gly           #   10                                                                        - CTG GTG CAG CCC TCA CAG ACC CTG TCC CTC AC - #C TGC ACT GTC TCT GGG          144                                                                          Leu Val Gln Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly           #                 25                                                          - TTC TCA TTA ACC AGC TAT ACT GTA AGC TGG GT - #T CGC CAG CCT CCA GGA          192                                                                          Phe Ser Leu Thr Ser Tyr Thr Val Ser Trp Va - #l Arg Gln Pro Pro Gly           #             40                                                              - AAG GGT CTG GAG TGG ATT GCA GCA ATA TCA AG - #T GGT GGA AGC ACA TAT          240                                                                          Lys Gly Leu Glu Trp Ile Ala Ala Ile Ser Se - #r Gly Gly Ser Thr Tyr           #         55                                                                  - TAT AAT TCA GCT CTC AAA TCA CGA CTG AGC AT - #C AGC AGG GAC ACC TCC          288                                                                          Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser Il - #e Ser Arg Asp Thr Ser           #     70                                                                      - AAG AGC CAA GTT TTC TTA AAA ATG AAC AGT CT - #G CAA ACT GAA GAC ACA          336                                                                          Lys Ser Gln Val Phe Leu Lys Met Asn Ser Le - #u Gln Thr Glu Asp Thr           # 90                                                                          - GCC ATG TAC TTC TGT GCC CCT TCT GAG GGG GC - #C TGG GGC CAA GGA GTC          384                                                                          Ala Met Tyr Phe Cys Ala Pro Ser Glu Gly Al - #a Trp Gly Gln Gly Val           #                105                                                          #                 405 TCA GAG                                                 Met Val Thr Val Ser Ser Glu                                                               110                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 402 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 19..78                                                          (C) IDENTIFICATION METHOD:                                                         BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN                 #CONSENSUS     ESTABLISHED                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 148..180                                                        (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABILSHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 1"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 226..246                                                        (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 2"                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 343..369                                                        (C) IDENTIFICATION METHOD: - # BY SIMILARITY                                       WITH KNOW - #N SEQUENCE OR TO AN ESTABLISHED                                  CONSENSUS                                                      #/product= "HYPERVARIABLE REGION 3"                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #TTA GGG CTG CTG       51 ATG GCT CCA GTC CAG CTC                             #Leu Leuet Ala Pro Val Gln Leu Leu Gly                                        10                                                                            - CTG ATT TGG CTC CCA GCC ATG AGA TGT GAC AT - #C CAG ATG ACC CAG TCT           99                                                                          Leu Ile Trp Leu Pro Ala Met Arg Cys Asp Il - #e Gln Met Thr Gln Ser           #1               5                                                            - CCT TCA TTC CTG TCT GCA TCT GTG GGA GAC AG - #A GTC ACT ATC AAC TGC          147                                                                          Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Ar - #g Val Thr Ile Asn Cys           #         20                                                                  - AAA GCA AGT CAG AAT ATT AAC AAG TAC TTA AA - #C TGG TAT CAG CAA AAG          195                                                                          Lys Ala Ser Gln Asn Ile Asn Lys Tyr Leu As - #n Trp Tyr Gln Gln Lys           #     35                                                                      - CTT GGA GAA GCT CCC AAA CGC CTG ATA TAT AA - #T ACA AAC AAT TTG CAA          243                                                                          Leu Gly Glu Ala Pro Lys Arg Leu Ile Tyr As - #n Thr Asn Asn Leu Gln           # 55                                                                          - ACG GGC ATT CCA TCA AGG TTC AGT GGC AGT GG - #A TCT GGT ACA GAT TAC          291                                                                          Thr Gly Ile Pro Ser Arg Phe Ser Gly Ser Gl - #y Ser Gly Thr Asp Tyr           #                 70                                                          - ACA CTC ACC ATC AGC AGC CTG CAG CCT GAA GA - #T TTT GCC ACA TAT TTC          339                                                                          Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu As - #p Phe Ala Thr Tyr Phe           #             85                                                              - TGC TTG CAG CAT AAT AGT TTT CCG AAC ACG TT - #T GGA GCT GGG ACC AAG          387                                                                          Cys Leu Gln His Asn Ser Phe Pro Asn Thr Ph - #e Gly Ala Gly Thr Lys           #        100                                                                  #   402            GG                                                         Leu Glu Leu Lys Arg                                                               105                                                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #    15            AC                                                         Asp Tyr Asn Met Asp                                                             1               5                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucliec acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - TAT ATT TAT CCT AAC AAT GGT GGT ACT GGC TA - #C AAC CAG AAG TTC AAG           48                                                                          Tyr Ile Tyr Pro Asn Asn Gly Gly Thr Gly Ty - #r Asn Gln Lys Phe Lys           #                 15                                                          #             51                                                              Ser                                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #         33T TAC TAC GGC TAC ATG TTT GCT TA - #C                             Tyr Gly His Tyr Tyr Gly Tyr Met Phe Ala Ty - #r                               #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #           30     GT GTA AGT TAC ATG CAC                                     Ser Ala Ser Ser Ser Val Ser Tyr Met His                                       #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #21                TG GCT TCT                                                 Ser Thr Ser Asn Leu Ala Ser                                                     1               5                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #             27   GT TAC CCG TAC ACG                                         Gln Gln Arg Ser Ser Tyr Pro Tyr Thr                                             1               5                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #          31      CA GCC TCC ACC AAG GGC C                                   Val Thr Val Ser Ala Ala Ser Thr Lys Gly                                       #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #AAA CGA ACT GTG GCT         46 CTG GAA ATA                                     Thr Phe Gly Gly Gly Thr Lys Leu Glu I - #le Lys Arg Thr Val Ala             # 15                                                                          #            51                                                               Ala                                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - CAA GGA GTC ATG GTC ACA GTC TCG AGC GCC TC - #C ACC AAG GGC                 #  42                                                                         Gln Gly Val Met Val Thr Val Ser Ser Ala Se - #r Thr Lys Gly                   #                 10                                                          #               43                                                            - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #AAA CGA ACT GTG GCT         46 CTT GAG CTC                                     Thr Phe Gly Ala Gly Thr Lys Leu Glu L - #eu Lys Arg Thr Val Ala             # 15                                                                          #            51                                                               Ala                                                                           - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 812 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (vi) ORIGINAL SOURCE:                                                             (B) STRAIN: HYBRIDOMA K - #M50                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: TATA.sub.-- - #signal                                           (B) LOCATION: 261..267                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - AAAGTCAGAC AACTTTGTAG AGTAGGTTCT ATCAATCCTA CTGCAATCCA AC - #ATCACTGA         60                                                                          - GGACAAATGT TTATACTGAG GAACCTGGTC TTGTGTGATA CGTACTTTCT GT - #GGGAAGCA        120                                                                          - GATACGCACT CTCATGTGGC TCCTGAATTT CCCATCACAG AATGATACAT CT - #TGAGTCCT        180                                                                          - AAAATTTAAG TACACCATCA GTGTCAGCAC CTGGTGAGGA AATGCAAATC TC - #TCCTGGAT        240                                                                          - CCACCCAACC TTGGGTTGAA AAGCCAAAGC TGGGCCTGGG TACTCACTGG TG - #TGCAGCC         299                                                                          - ATG GAC AGG CTT ACT TCC TCA TTC CTA CTG CT - #G ATG GTC CCT GCA              34 - #4                                                                      Met Asp Arg Leu Thr Ser Ser Phe Leu Leu Le - #u Met Val Pro Ala               - TGTGAGTACC AAAGCTTCCT AAGTGATGAA CTGTTCTATC CTCACCTGTT CA - #AACCTGAC        404                                                                          - CTCCTCCCCT TTGATTTCTC CACAG AT GTC CTG TCT CAG G - #TT ACT CTG AAA           455                                                                          #Val Thr Leu Lysl Leu Ser Gln                                                 #      5  1                                                                   - GAA TCT GGC CCT GGG ATA TTG CAG CCC TCC CA - #G ACC CTC AGT CTG ACT          503                                                                          Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gl - #n Thr Leu Ser Leu Thr           #                 20                                                          - TGC TCT TTC TCT GGG TTT TCA CTG AGC ACT TA - #T GGT ATG TGT GTG GGC          551                                                                          Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ty - #r Gly Met Cys Val Gly           #             35                                                              - TGG ATT CGT CAG TCT TCA GGG AAG GGT CTG GA - #G TGG CTG GCA AAC GTT          599                                                                          Trp Ile Arg Gln Ser Ser Gly Lys Gly Leu Gl - #u Trp Leu Ala Asn Val           #         50                                                                  - TGG TGG AGT GAT GCT AAG TAC TAC AAT CCA TC - #T CTG AAA AAC CGG CTC          647                                                                          Trp Trp Ser Asp Ala Lys Tyr Tyr Asn Pro Se - #r Leu Lys Asn Arg Leu           #     65                                                                      - ACA ATC TCC AAG GAC ACC TCC AAC AAC CAA GC - #A TTC CTC AAG ATC ACC          695                                                                          Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Al - #a Phe Leu Lys Ile Thr           # 85                                                                          - AAT ATG GAC ACT GCA GAT ACT GCC ATA TAC TA - #C TGT GCT GGG AGA GGG          743                                                                          Asn Met Asp Thr Ala Asp Thr Ala Ile Tyr Ty - #r Cys Ala Gly Arg Gly           #                100                                                          - GCT ACG GAG GGT ATA GTG AGC TTT GAT TAC TG - #G GGC CAC GGA GTC ATG          791                                                                          Ala Thr Glu Gly Ile Val Ser Phe Asp Tyr Tr - #p Gly His Gly Val Met           #           115                                                               #                 812 GGTAAG                                                  Val Thr Val Ser Ser                                                                   120                                                                   - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #                 46ATA TCAAGCTTGT CGACTCTAGA GGTACC                          - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #            29    GCAG CCACAGTTC                                             - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 408 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (vi) ORIGINAL SOURCE:                                                             (B) STRAIN: HYBRIDOMA K - #M-641                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 25..84                                                          (C) IDENTIFICATION METHOD:                                                         BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN                 #CONSENSUS     ESTABLISHED                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - AATTCGGCAC GAGTCAGCCT GGAC ATG ATG TCC TCT GCT C - #AG TTC CTT GGT            51                                                                          #Gln Phe Leu Glyt Ser Ser Ala                                                 15                                                                            - CTC CTG TTG CTC TGT TTT CAA GGT ACC AGA TG - #T GAT ATC CAG ATG ACA           99                                                                          Leu Leu Leu Leu Cys Phe Gln Gly Thr Arg Cy - #s Asp Ile Gln Met Thr           #  5  1                                                                       - CAG ACT GCA TCC TCC CTG CCT GCC TCT CTG GG - #A GAC AGA GTC ACC ATC          147                                                                          Gln Thr Ala Ser Ser Leu Pro Ala Ser Leu Gl - #y Asp Arg Val Thr Ile           #                 20                                                          - AGT TGC AGT GCA AGT CAG GAC ATT AGT AAT TA - #T TTA AAC TGG TAT CAA          195                                                                          Ser Cys Ser Ala Ser Gln Asp Ile Ser Asn Ty - #r Leu Asn Trp Tyr Gln           #             35                                                              - CAG AAA CCA GAT GGA ACT GTT AAA CTC CTG AT - #C TTT TAC TCA TCA AAT          243                                                                          Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Il - #e Phe Tyr Ser Ser Asn           #         50                                                                  - TTA CAC TCG GGA GTC CCA TCA AGG TTC AGT GG - #C GGT GGG TCC GGG ACA          291                                                                          Leu His Ser Gly Val Pro Ser Arg Phe Ser Gl - #y Gly Gly Ser Gly Thr           #     65                                                                      - GAT TAT TCT CTC ACC ATC AGC AAC CTG GAG CC - #T GAA GAT ATT GCC ACT          339                                                                          Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pr - #o Glu Asp Ile Ala Thr           # 85                                                                          - TAC TTT TGT CAT CAG TAT AGT AAG CTT CCG TG - #G ACG TCC GGT GGA GGC          387                                                                          Tyr Phe Cys His Gln Tyr Ser Lys Leu Pro Tr - #p Thr Ser Gly Gly Gly           #                100                                                          #                 408 AAA CGG                                                 Thr Lys Leu Glu Ile Lys Arg                                                               105                                                               - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 403 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (vi) ORIGINAL SOURCE:                                                             (B) STRAIN: HYBRIDOMA K - #M-641                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 14..43                                                          (C) IDENTIFICATION METHOD:                                                         BY SIMILA - #RITY WITH KNOWN SEQUENCE OR TO AN                 #CONSENSUS     ESTABLISHED                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - AATTCGGCAC GAG CTT GTC CTT GTT TTC AAA GGT GT - #T CAG TGT GAA GTG            49                                                                          #Leu Val Phe Lys Gly Val Gln Cys Glu Val                                      #                1                                                            - ACG CTG GTG GAG TCT GGG GGA GAC TTT GTG AA - #A CCT GGA GGG TCC CTG           97                                                                          Thr Leu Val Glu Ser Gly Gly Asp Phe Val Ly - #s Pro Gly Gly Ser Leu           #          15                                                                 - AAA GTC TCC TGT GCA GCC TCT GGA TTC GCT TT - #C AGT CAT TAT GCC ATG          145                                                                          Lys Val Ser Cys Ala Ala Ser Gly Phe Ala Ph - #e Ser His Tyr Ala Met           #     30                                                                      - TCT TGG GTT CGC CAG ACT CCG GCG AAG AGG CT - #G GAA TGG GTC GCA TAT          193                                                                          Ser Trp Val Arg Gln Thr Pro Ala Lys Arg Le - #u Glu Trp Val Ala Tyr           # 50                                                                          - ATT AGT AGT GGT GGT AGT GGC ACC TAC TAT TC - #A GAC AGT GTA AAG GGC          241                                                                          Ile Ser Ser Gly Gly Ser Gly Thr Tyr Tyr Se - #r Asp Ser Val Lys Gly           #                 65                                                          - CGA TTC ACC ATT TCC AGA GAC AAT GCC AAG AA - #C ACC CTG TAC C TG CAA         289                                                                          Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys As - #n Thr Leu Tyr Leu Gln           #             80                                                              - ATG CGC AGT CTG AGG TCT GAG GAC TCG GCC AT - #G TAT TTC TGT ACA AGA          337                                                                          Met Arg Ser Leu Arg Ser Glu Asp Ser Ala Me - #t Tyr Phe Cys Thr Arg           #         95                                                                  - GTT AAA CTG GGA ACC TAC TAC TTT GAC TCC TG - #G GGC CAA GGC ACC ACT          385                                                                          Val Lys Leu Gly Thr Tyr Tyr Phe Asp Ser Tr - #p Gly Gln Gly Thr Thr           #   110                                                                       # 403              CA GCT                                                     Leu Thr Val Ser Ser Ala                                                       115                 1 - #20                                                   - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #       35         TTT GGG CTC AGC TGG CTT TTT                                         Met Glu Phe Gly Leu S - #er Trp Leu Phe                              #        5 1                                                                  - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - CAA GGT ACC ACG TTA ACT GTC TCC TCA GCC TC - #C ACC AAG GGC                 #  42                                                                         Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Se - #r Thr Lys Gly                   #                 10                                                          #               43                                                            - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 80 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - GGCCGCACCA TGGGATGGAG CTGGATCTTT CTCTTCCTCC TGTCAGGAAC TG - #CTGGTGTC         60                                                                          # 80               TGCA                                                       - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 59 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - GGAGAGCGGT CCAGGTCTTG TGAGGCCTAG CCAGACCCTG AGCCTGACCT GC - #ACCGTGT          59                                                                          - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - CCGGATTCAC CTTCAGCGAC TACAACATGG ACTGGGTGAG ACAGCCACCT GG - #ACGAGGTC         60                                                                          - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 81 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - TCGAGTGGAT TGGATATATT TATCCTAACA ATGGTGGTAC TGGCTACAAC CA - #GAAGTTCA         60                                                                          #81                GCTG G                                                     - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 61 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - TCGACACCAG CAAGAACCAG TTCAGCCTGA GACTCAGCAG CGTGACAGCC GC - #CGACACCG         60                                                                          #               61                                                            - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - GGTCTATTAT TGTGCGCGCT ACGGTCATTA CTACGGCTAC ATGTTTGCTT AC - #TGGGGTCA         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #       35         TCAG CCTCCACCAA GGGCC                                      - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 77 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - AATTCACCAT GCATTTTCAA GTGCAGATTT TCAGCTTCCT GCTAATCAGT GC - #CTCAGTCA         60                                                                          #   77             T                                                          - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 62 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - ATCCAGCTGA CCCAGAGCCC AAGCAGCCTG AGCGCTAGCG TGGGTGACAG AG - #TGACCATG         60                                                                          #              62                                                             - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 65 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - GTGCAGTGCC AGCTCAAGTG TAAGTTACAT GCACTGGTAT CAGCAGAAGC CA - #GGTAAGGC         60                                                                          #            65                                                               - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #45                CACA TCCAACCTGG CTTCTGGTGT GCCAT                           - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 76 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 #ID NO:34:(xi) SEQUENCE DESCRIPTION: SEQ                                      - CTAGATTCAG CGGTAGCGGT AGCGGTACAG ACTTCACCTT CACCATCAGC AG - #CCTCCAGC         60                                                                          #    76                                                                       - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 84 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                #synthetic DNAECULE TYPE: other nucleic acid,                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - GTACTACTGC CAGCAAAGGA GTAGTTACCC GTACACGTTC GGCGGGGGGA CC - #AAGGTGGA         60                                                                          #                84GCTG CACC                                                  - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 442 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - GGCCGCACC ATG GGA TGG AGC TGG ATC TTT CTC TTC - # CTC CTG TCA GGA             48                                                                          #Ile Phe Leu Phe Leu Leu Ser Gly                                              10                                                                            - ACT GCT GGT GTC CTC TCT CAG GTC CAA CTG CA - #G GAG AGC GGT CCA GGT           96                                                                          Thr Ala Gly Val Leu Ser Gln Val Gln Leu Gl - #n Glu Ser Gly Pro Gly           #   10                                                                        - CTT GTG AGG CCT AGC CAG ACC CTG AGC CTG AC - #C TGC ACC GTG TCC GGA          144                                                                          Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly           #                 25                                                          - TTC ACC TTC AGC GAC TAC AAC ATG GAC TGG GT - #G AGA CAG CCA CCT GGA          192                                                                          Phe Thr Phe Ser Asp Tyr Asn Met Asp Trp Va - #l Arg Gln Pro Pro Gly           #             40                                                              - CGA GGT CTC GAG TGG ATT GGA TAT ATT TAT CC - #T AAC AAT GGT GGT ACT          240                                                                          Arg Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pr - #o Asn Asn Gly Gly Thr           #         55                                                                  - GGC TAC AAC CAG AAG TTC AAG AGC AGA GTG AC - #A ATG CTG GTC GAC ACC          288                                                                          Gly Tyr Asn Gln Lys Phe Lys Ser Arg Val Th - #r Met Leu Val Asp Thr           #     70                                                                      - AGC AAG AAC CAG TTC AGC CTG AGA CTC AGC AG - #C GTG ACA GCC GCC GAC          336                                                                          Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Se - #r Val Thr Ala Ala Asp           # 90                                                                          - ACC GCG GTC TAT TAT TGT GCG CGC TAC GGT CA - #T TAC TAC GGC TAC ATG          384                                                                          Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Gly Hi - #s Tyr Tyr Gly Tyr Met           #                105                                                          - TTT GCT TAC TGG GGT CAA GGT ACC ACC GTC AC - #A GTC TCC TCA GCC TCC          432                                                                          Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Ala Ser           #           120                                                               #       442                                                                   Thr Lys Gly                                                                           125                                                                   - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 409 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #TTC CTG CTA ATC AGT      50TG CAG ATT TTC AGC                                         Met His Phe Gln Val G - #ln Ile Phe Ser Phe Leu Leu Ile Ser          10                                                                            - GCC TCA GTC ATA ATG TCC AGA GGA GAT ATC CA - #G CTG ACC CAG AGC CCA           98                                                                          Ala Ser Val Ile Met Ser Arg Gly Asp Ile Gl - #n Leu Thr Gln Ser Pro           #           5  1                                                              - AGC AGC CTG AGC GCT AGC GTG GGT GAC AGA GT - #G ACC ATC ACG TGC AGT          146                                                                          Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Va - #l Thr Ile Thr Cys Ser           #     20                                                                      - GCC AGC TCA AGT GTA AGT TAC ATG CAC TGG TA - #T CAG CAG AAG CCA GGT          194                                                                          Ala Ser Ser Ser Val Ser Tyr Met His Trp Ty - #r Gln Gln Lys Pro Gly           # 40                                                                          - AAG GCT CCA AAG CTT CTG ATC TAC AGC ACA TC - #C AAC CTG GCT TCT GGT          242                                                                          Lys Ala Pro Lys Leu Leu Ile Tyr Ser Thr Se - #r Asn Leu Ala Ser Gly           #                 55                                                          - GTG CCA TCT AGA TTC AGC GGT AGC GGT AGC GG - #T ACA GAC TTC ACC TTC          290                                                                          Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gl - #y Thr Asp Phe Thr Phe           #             70                                                              - ACC ATC AGC AGC CTC CAG CCA GAG GAC ATC GC - #T ACG TAC TAC TGC CAG          338                                                                          Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Al - #a Thr Tyr Tyr Cys Gln           #         85                                                                  - CAA AGG AGT AGT TAC CCG TAC ACG TTC GGC GG - #G GGG ACC AAG GTG GAA          386                                                                          Gln Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Gl - #y Gly Thr Lys Val Glu           #    100                                                                      #               409TG GCT GCA CC                                              Ile Lys Arg Thr Val Ala Ala                                                   105                 1 - #10                                                   - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                #          32      AGGA GCAGGTGAAT TC                                         - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                #    40            TCCT CAGTTAACAC TGAGTGGTAC                                 - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #21                GCAC C                                                     - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              #ID NO:41:(xi) SEQUENCE DESCRIPTION: SEQ                                      #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #              26  CCGC GGCCGC                                                - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                #        34        CCAC TAGTCGCGAG GTAC                                       - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                # 20               CCCG                                                       - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                # 20               CCAC                                                       - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #       36         GGTT CGATAAATCG ATACCG                                     - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                #    40            AACC GTACGAAGAA TTCATGAGCT                                 - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #       35         ACCA AGCTGGAAAT AAAAC                                      - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                #       35         GCTT GGTCCCCCCT CCGAA                                      - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 61 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                - TCGACACCAG CAAGAACACA GCCTACCTGA GACTCAGCAG CGTGACAGCC GC - #CGACACCG         60                                                                          #               61                                                            - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 59 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                - CCGGATACAC ATTCACTGAC TACAACATGG ACTGGGTGAG ACAGAGCCAT GA - #CGAGGTC          59                                                                          - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 442 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 19..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensus                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 31..35                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 50..66                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 99..109                                                         (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                - GGCCGCACC ATG GGA TGG AGC TGG ATC TTT CTC TTC - # CTC CTG TCA GGA             48                                                                          #Ile Phe Leu Phe Leu Leu Ser Gly                                              10                                                                            - ACT GCT GGT GTC CTC TCT CAG GTC CAA CTG CA - #G GAG AGC GGT CCA GGT           96                                                                          Thr Ala Gly Val Leu Ser Gln Val Gln Leu Gl - #n Glu Ser Gly Pro Gly           #   10                                                                        - CTT GTG AGG CCT AGC CAG ACC CTG AGC CTG AC - #C TGC ACC GTG TCC GGA          144                                                                          Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly           #                 25                                                          - TTC ACC TTC AGC GAC TAC AAC ATG GAC TGG GT - #G AGA CAG CCA CCT GGA          192                                                                          Phe Thr Phe Ser Asp Tyr Asn Met Asp Trp Va - #l Arg Gln Pro Pro Gly           #             40                                                              - CGA GGT CTC GAG TGG ATT GGA TAT ATT TAT CC - #T AAC AAT GGT GGT ACT          240                                                                          Arg Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pr - #o Asn Asn Gly Gly Thr           #         55                                                                  - GGC TAC AAC CAG AAG TTC AAG AGC AGA GTG AC - #A ATG CTG GTC GAC ACC          288                                                                          Gly Tyr Asn Gln Lys Phe Lys Ser Arg Val Th - #r Met Leu Val Asp Thr           #     70                                                                      - AGC AAG AAC ACA GCC TAC CTG AGA CTC AGC AG - #C GTG ACA GCC GCC GAC          336                                                                          Ser Lys Asn Thr Ala Tyr Leu Arg Leu Ser Se - #r Val Thr Ala Ala Asp           # 90                                                                          - ACC GCG GTC TAT TAT TGT GCA ACC TAC GGT CA - #T TAC TAC GGC TAC ATG          384                                                                          Thr Ala Val Tyr Tyr Cys Ala Thr Tyr Gly Hi - #s Tyr Tyr Gly Tyr Met           #                105                                                          - TTT GCT TAC TGG GGT CAA GGT ACC ACC GTC AC - #A GTC TCC TCA GCC TCC          432                                                                          Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Ala Ser           #           120                                                               #       442                                                                   Thr Lys Gly                                                                           125                                                                   - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 442 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 19..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 31..35                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 50..66                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 99..109                                                         (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                - GGCCGCACC ATG GGA TGG AGC TGG ATC TTT CTC TTC - # CTC CTG TCA GGA             48                                                                          #Ile Phe Leu Phe Leu Leu Ser Gly                                              10                                                                            - ACT GCT GGT GTC CTC TCT CAG GTC CAA CTG CA - #G GAG AGC GGT CCA GGT           96                                                                          Thr Ala Gly Val Leu Ser Gln Val Gln Leu Gl - #n Glu Ser Gly Pro Gly           #   10                                                                        - CTT GTG AGG CCT AGC CAG ACC CTG AGC CTG AC - #C TGC ACC GTG TCC GGA          144                                                                          Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Th - #r Cys Thr Val Ser Gly           #                 25                                                          - TAC ACA TTC ACT GAC TAC AAC ATG GAC TGG GT - #G AGA CAG AGC CAT GGA          192                                                                          Tyr Thr Phe Thr Asp Tyr Asn Met Asp Trp Va - #l Arg Gln Ser His Gly           #             40                                                              - CGA GGT CTC GAG TGG ATT GGA TAT ATT TAT CC - #T AAC AAT GGT GGT ACT          240                                                                          Arg Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pr - #o Asn Asn Gly Gly Thr           #         55                                                                  - GGC TAC AAC CAG AAG TTC AAG AGC AGA GTG AC - #A ATG CTG GTC GAC ACC          288                                                                          Gly Tyr Asn Gln Lys Phe Lys Ser Arg Val Th - #r Met Leu Val Asp Thr           #     70                                                                      - AGC AAG AAC CAG TTC AGC CTG AGA CTC AGC AG - #C GTG ACA GCC GCC GAC          336                                                                          Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Se - #r Val Thr Ala Ala Asp           # 90                                                                          - ACC GCG GTC TAT TAT TGT GCA ACC TAC GGT CA - #T TAC TAC GGC TAC ATG          384                                                                          Thr Ala Val Tyr Tyr Cys Ala Thr Tyr Gly Hi - #s Tyr Tyr Gly Tyr Met           #                105                                                          - TTT GCT TAC TGG GGT CAA GGT ACC ACC GTC AC - #A GTC TCC TCA GCC TCC          432                                                                          Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Ala Ser           #           120                                                               #       442                                                                   Thr Lys Gly                                                                           125                                                                   - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 100 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                - CAGGAAACAG CTATGACGCG GCCGCCACCA TGGGATGGAG CTGGATCTTT CT - #CTTCCTCC         60                                                                          #   100            TGTC CTCTCTGAGG TGCAGCTGGT                                 - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 100 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                - AGTCAGTGAA GGTGTATCCG GAAGCCTTGC AGGAGACCTT CACTGAGGCC CC - #AGGCTTCT         60                                                                          #   100            CTGC ACCAGCTGCA CCTCAGAGAG                                 - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 100 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                - CGGATACACC TTCACTGACT ACAACATGGA CTGGGTGCGA CAGGCCCCTG GA - #CAAGGGCT         60                                                                          #   100            ATTT ATCCTAACAA TGGTGGTACT                                 - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 94 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                - AGCTCCATGT AGGCTGTGCT CGTGGATGTG TCTACGGTAA TGGTGACCTT GC - #TCTTGAAC         60                                                                          #        94        TACC ACCATTGTTA GGAT                                       - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 96 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                - AGCACAGCCT ACATGGAGCT GCACAGCCTG AGATCTGAGG ACACGGCCGT GT - #ATTACTGT         60                                                                          #       96         ACTA CGGCTACATG TTTGCT                                     - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 90 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                - GTTTTCCCAG TCACGACGGG CCCTTGGTGG AGGCTGAGGA GACGGTGACC AG - #GGTTCCCT         60                                                                          #           90     CATG TAGCCGTAGT                                            - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 432 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 19..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 31..35                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 50..66                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 99..109                                                         (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                - ATG GGA TGG AGC TGG ATC TTT CTC TTC CTC CT - #G TCA GGA ACT GCA GGT           48                                                                          Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Le - #u Ser Gly Thr Ala Gly           5                                                                             - GTC CTC TCT GAG GTG CAG CTG GTG CAG TCT GG - #A GCA GAG GTG AAG AAG           96                                                                          Val Leu Ser Glu Val Gln Leu Val Gln Ser Gl - #y Ala Glu Val Lys Lys           #           10                                                                - CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GC - #T TCC GGA TAC ACC TTC          144                                                                          Pro Gly Ala Ser Val Lys Val Ser Cys Lys Al - #a Ser Gly Tyr Thr Phe           #     25                                                                      - ACT GAC TAC AAC ATG GAC TGG GTG CGA CAG GC - #C CCT GGA CAA GGG CTC          192                                                                          Thr Asp Tyr Asn Met Asp Trp Val Arg Gln Al - #a Pro Gly Gln Gly Leu           # 45                                                                          - GAG TGG ATG GGA TAT ATT TAT CCT AAC AAT GG - #T GGT ACT GGC TAC AAC          240                                                                          Glu Trp Met Gly Tyr Ile Tyr Pro Asn Asn Gl - #y Gly Thr Gly Tyr Asn           #                 60                                                          - CAG AAG TTC AAG AGC AAG GTC ACC ATT ACC GT - #A GAC ACA TCC ACG AGC          288                                                                          Gln Lys Phe Lys Ser Lys Val Thr Ile Thr Va - #l Asp Thr Ser Thr Ser           #             75                                                              - ACA GCC TAC ATG GAG CTG CAC AGC CTG AGA TC - #T GAG GAC ACG GCC GTG          336                                                                          Thr Ala Tyr Met Glu Leu His Ser Leu Arg Se - #r Glu Asp Thr Ala Val           #         90                                                                  - TAT TAC TGT GCG ACC TAC GGT CAT TAC TAC GG - #C TAC ATG TTT GCT TAC          384                                                                          Tyr Tyr Cys Ala Thr Tyr Gly His Tyr Tyr Gl - #y Tyr Met Phe Ala Tyr           #    105                                                                      - TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TC - #A GCC TCC ACC AAG GGC          432                                                                          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Se - #r Ala Ser Thr Lys Gly           110                 1 - #15                 1 - #20                 1 -       #25                                                                           - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                - GTACTACTGC CAGCAAAGGA GTAGTTACCC GTACACGTTC GGCGGGGGGA CC - #AAGGTGGA         60                                                                          #          68                                                                 - (2) INFORMATION FOR SEQ ID NO:62:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                #               25 TAGC GCTCA                                                 - (2) INFORMATION FOR SEQ ID NO:63:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                #               25 TGAC AGAGT                                                 - (2) INFORMATION FOR SEQ ID NO:64:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT CTG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA GA - #C TTC ACC TTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Phe Thr Ile           #     70                                                                      - AGC AGC CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:65:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                #               25 GCTT TGGAG                                                 - (2) INFORMATION FOR SEQ ID NO:66:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                #               25 CTAC AGCAC                                                 - (2) INFORMATION FOR SEQ ID NO:67:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC GTG GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Val Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA GA - #C TTC ACC TTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Phe Thr Ile           #     70                                                                      - AGC AGC CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:68:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                #               25 AGCC TGGAG                                                 - (2) INFORMATION FOR SEQ ID NO:69:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                #               25 CTGC TACGT                                                 - (2) INFORMATION FOR SEQ ID NO:70:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #an established consensu                                                      #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC GTG GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Val Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT CTG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA GA - #C TTC ACC TTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Phe Thr Ile           #     70                                                                      - AGC AGC CTC CAG GCT GAA GAT GCT GCT ACG TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Ser Leu Gln Ala Glu Asp Ala Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:71:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                #               25 ATGT ACCGC                                                 - (2) INFORMATION FOR SEQ ID NO:72:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                #               25 TTTC ACCAT                                                 - (2) INFORMATION FOR SEQ ID NO:73:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT TTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Phe Thr Ile           #     70                                                                      - AGC AGC CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:74:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                #    40            TGAT GGTGAAAGAG TAAGATGTAC                                 - (2) INFORMATION FOR SEQ ID NO:75:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                #    40            CACC ATCAGCCGAA TGGAGCCAGA                                 - (2) INFORMATION FOR SEQ ID NO:76:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT TTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Phe Thr Ile           #     70                                                                      - AGC CGA ATG GAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Arg Met Glu Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:77:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                # 20               GCAT                                                       - (2) INFORMATION FOR SEQ ID NO:78:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                # 20               AGAA                                                       - (2) INFORMATION FOR SEQ ID NO:79:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TTC CAG CA - #G AAG CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Phe Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT TTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Phe Thr Ile           #     70                                                                      - AGC AGC CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:80:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                #               25 TGAG AGAGT                                                 - (2) INFORMATION FOR SEQ ID NO:81:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                #               25 CCGA CTCCA                                                 - (2) INFORMATION FOR SEQ ID NO:82:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TAT CAG CA - #G AAG CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT CTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Leu Thr Ile           #     70                                                                      - AGC CGA CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Arg Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:83:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TTC CAG CA - #G AAG CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Phe Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT CTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Leu Thr Ile           #     70                                                                      - AGC CGA CTC CAG CCA GAG GAC ATC GCT ACG TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Arg Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:84:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 94 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                - CAGGAAACAG CTATGACGAA TTCCACCATG CATTTTCAAG TGCAGATTTT CA - #GCTTCCTG         60                                                                          #        94        TCAT AATGTCCAGA GGAG                                       - (2) INFORMATION FOR SEQ ID NO:85:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 88 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                - ACAAGTGATG GTGACTCTGT CTCCTGGAGA TGCAGACATG GAGGATGGAG AC - #TGGGTCAG         60                                                                          #             88   GACA TTATGACT                                              - (2) INFORMATION FOR SEQ ID NO:86:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 92 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                - ACAGAGTCAC CATCACTTGT AGTGCAAGTT CAAGTGTAAG TTACATGCAC TG - #GTTTCAGC         60                                                                          #          92      ACCT AAGCTCTGGA TC                                         - (2) INFORMATION FOR SEQ ID NO:87:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 87 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                - AAGATGTACC GCTACCGCTA CCGCTGAATC TAGATGGCAC ACCAGAAGCT AA - #ATTTGAAG         60                                                                          #             87   CTTA GGTGATT                                               - (2) INFORMATION FOR SEQ ID NO:88:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 89 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                - TAGCGGTAGC GGTACATCTT ACTCTCTCAC CATCAGCAGC ATGCAGCCTG AA - #GATTTTGC         60                                                                          #            89    CAAA GGAGTAGTT                                             - (2) INFORMATION FOR SEQ ID NO:89:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 84 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                - GTTTTCCCAG TCACGACCGT ACGTTTGATT TCCAGCTTGG TCCCCTGGCC GA - #ACGTGTAC         60                                                                          #                84GCTG ACAG                                                  - (2) INFORMATION FOR SEQ ID NO:90:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens and mouse                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                 22..-1    (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR1"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR2"ER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: - # by similarity with known             sequence                                                                      #established consensun                                                        #/product= "CDR3"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAC ATC CAG CTG AC - #C CAG TCT CCA TCC TCC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - ATG TCT GCA TCT CCA GGA GAC AGA GTC ACC AT - #C ACT TGT AGT GCA AGT          144                                                                          Met Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TTT CAG CA - #G AAA CCA GGG AAA TCA          192                                                                          Ser Ser Val Ser Tyr Met His Trp Phe Gln Gl - #n Lys Pro Gly Lys Ser           #             40                                                              - CCT AAG CTC TGG ATC TAC TCA ACT TCA AAT TT - #A GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - TCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT CTC ACC ATC          288                                                                          Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Leu Thr Ile           #     70                                                                      - AGC AGC ATG CAG CCT GAA GAT TTT GCA ACT TA - #T TAC TGT CAG CAA AGG          336                                                                          Ser Ser Met Gln Pro Glu Asp Phe Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC CAG GGG AC - #C AAG CTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Th - #r Lys Leu Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       - (2) INFORMATION FOR SEQ ID NO:91:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                #       35         AGGG CTCTGCTTCA CCCAG                                      - (2) INFORMATION FOR SEQ ID NO:92:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                #       35         CCTG GAAAGAGCCT CGAGT                                      - (2) INFORMATION FOR SEQ ID NO:93:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 433 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 1..57                                                 #/note= "IDENTIFICATION METHOD: by                                            #with a known sequence or to an established                                   #sequence"     consensus                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 148..162                                              #/product= "hypervariable region 1"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 205..255                                              #/product= "hypervariable region 2"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 352..384                                              #/product= "hypervariable region 3"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                - ATG GGA TGG AGC TGG ATC TTT CTC TTC CTC CT - #G TCA GGA ACT GCA GGT           48                                                                          Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Le - #u Ser Gly Thr Ala Gly           5                                                                             - GTC CTC TCT GAG GTG CAG CTG GTG CAG TCT GG - #A GCA GAG GTG AAG AAG           96                                                                          Val Leu Ser Glu Val Gln Leu Val Gln Ser Gl - #y Ala Glu Val Lys Lys           #           10                                                                - CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GC - #T TCC GGA TAC ACC TTC          144                                                                          Pro Gly Ala Ser Val Lys Val Ser Cys Lys Al - #a Ser Gly Tyr Thr Phe           #     25                                                                      - ACT GAC TAC AAC ATG GAC TGG GTG AAG CAG AG - #C CCT GGA AAG AGC CTC          192                                                                          Thr Asp Tyr Asn Met Asp Trp Val Lys Gln Se - #r Pro Gly Lys Ser Leu           # 45                                                                          - GAG TGG ATG GGA TAT ATT TAT CCT AAC AAT GG - #T GGT ACT GGC TAC AAC          240                                                                          Glu Trp Met Gly Tyr Ile Tyr Pro Asn Asn Gl - #y Gly Thr Gly Tyr Asn           #                 60                                                          - CAG AAG TTC AAG AGC AAG GTC ACC ATT ACC GT - #A GAC ACA TCC ACG AGC          288                                                                          Gln Lys Phe Lys Ser Lys Val Thr Ile Thr Va - #l Asp Thr Ser Thr Ser           #             75                                                              - ACA GCC TAC ATG GAG CTG CAC AGC CTG AGA TC - #T GAG GAC ACG GCC GTG          336                                                                          Thr Ala Tyr Met Glu Leu His Ser Leu Arg Se - #r Glu Asp Thr Ala Val           #         90                                                                  - TAT TAC TGT GCG ACC TAC GGT CAT TAC TAC GG - #C TAC ATG TTT GCT TAC          384                                                                          Tyr Tyr Cys Ala Thr Tyr Gly His Tyr Tyr Gl - #y Tyr Met Phe Ala Tyr           #    105                                                                      - TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TC - #A GCC TCC ACC AAG GGC          432                                                                          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Se - #r Ala Ser Thr Lys Gly           110                 1 - #15                 1 - #20                 1 -       #25                                                                           #              433                                                            - (2) INFORMATION FOR SEQ ID NO:94:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                #                24TCAC CCAG                                                  - (2) INFORMATION FOR SEQ ID NO:95:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                #                24CCTG GACA                                                  - (2) INFORMATION FOR SEQ ID NO:96:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 433 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 1..57                                                 #/note= "IDENTIFICATION METHOD: by                                            #with a known sequence or to an established                                   #sequence"     consensus                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 148..162                                              #/product= "hypervariable region 1"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 205..255                                              #/product= "hypervariable region 2"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 352..384                                              #/product= "hypervariable region 3"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                - ATG GGA TGG AGC TGG ATC TTT CTC TTC CTC CT - #G TCA GGA ACT GCA GGT           48                                                                          Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Le - #u Ser Gly Thr Ala Gly           5                                                                             - GTC CTC TCT GAG GTG CAG CTG GTG CAG TCT GG - #A GCA GAG GTG AAG AAG           96                                                                          Val Leu Ser Glu Val Gln Leu Val Gln Ser Gl - #y Ala Glu Val Lys Lys           #           10                                                                - CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GC - #T TCC GGA TAC ACC TTC          144                                                                          Pro Gly Ala Ser Val Lys Val Ser Cys Lys Al - #a Ser Gly Tyr Thr Phe           #     25                                                                      - ACT GAC TAC AAC ATG GAC TGG GTG AAG CAG AG - #C CCT GGA CAA GGG CTC          192                                                                          Thr Asp Tyr Asn Met Asp Trp Val Lys Gln Se - #r Pro Gly Gln Gly Leu           # 45                                                                          - GAG TGG ATG GGA TAT ATT TAT CCT AAC AAT GG - #T GGT ACT GGC TAC AAC          240                                                                          Glu Trp Met Gly Tyr Ile Tyr Pro Asn Asn Gl - #y Gly Thr Gly Tyr Asn           #                 60                                                          - CAG AAG TTC AAG AGC AAG GTC ACC ATT ACC GT - #A GAC ACA TCC ACG AGC          288                                                                          Gln Lys Phe Lys Ser Lys Val Thr Ile Thr Va - #l Asp Thr Ser Thr Ser           #             75                                                              - ACA GCC TAC ATG GAG CTG CAC AGC CTG AGA TC - #T GAG GAC ACG GCC GTG          336                                                                          Thr Ala Tyr Met Glu Leu His Ser Leu Arg Se - #r Glu Asp Thr Ala Val           #         90                                                                  - TAT TAC TGT GCG ACC TAC GGT CAT TAC TAC GG - #C TAC ATG TTT GCT TAC          384                                                                          Tyr Tyr Cys Ala Thr Tyr Gly His Tyr Tyr Gl - #y Tyr Met Phe Ala Tyr           #    105                                                                      - TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TC - #A GCC TCC ACC AAG GGC          432                                                                          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Se - #r Ala Ser Thr Lys Gly           110                 1 - #15                 1 - #20                 1 -       #25                                                                           #              433                                                            - (2) INFORMATION FOR SEQ ID NO:97:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                #               25 CCTT GCTCT                                                 - (2) INFORMATION FOR SEQ ID NO:98:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                #               25 GACC GTAGA                                                 - (2) INFORMATION FOR SEQ ID NO:99:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 433 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 1..57                                                 #/note= "IDENTIFICATION METHOD: by                                            #with a known sequence or to an established                                   #sequence"     consensus                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 148..162                                              #/product= "hypervariable region 1"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 205..255                                              #/product= "hypervariable region 2"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 352..384                                              #/product= "hypervariable region 3"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                - ATG GGA TGG AGC TGG ATC TTT CTC TTC CTC CT - #G TCA GGA ACT GCA GGT           48                                                                          Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Le - #u Ser Gly Thr Ala Gly           5                                                                             - GTC CTC TCT GAG GTG CAG CTG GTG CAG TCT GG - #A GCA GAG GTG AAG AAG           96                                                                          Val Leu Ser Glu Val Gln Leu Val Gln Ser Gl - #y Ala Glu Val Lys Lys           #           10                                                                - CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GC - #T TCC GGA TAC ACC TTC          144                                                                          Pro Gly Ala Ser Val Lys Val Ser Cys Lys Al - #a Ser Gly Tyr Thr Phe           #     25                                                                      - ACT GAC TAC AAC ATG GAC TGG GTG CGA CAG GC - #C CCT GGA CAA GGG CTC          192                                                                          Thr Asp Tyr Asn Met Asp Trp Val Arg Gln Al - #a Pro Gly Gln Gly Leu           # 45                                                                          - GAG TGG ATG GGA TAT ATT TAT CCT AAC AAT GG - #T GGT ACT GGC TAC AAC          240                                                                          Glu Trp Met Gly Tyr Ile Tyr Pro Asn Asn Gl - #y Gly Thr Gly Tyr Asn           #                 60                                                          - CAG AAG TTC AAG AGC AAG GCC ACC TTG ACC GT - #A GAC ACA TCC ACG AGC          288                                                                          Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Va - #l Asp Thr Ser Thr Ser           #             75                                                              - ACA GCC TAC ATG GAG CTG CAC AGC CTG AGA TC - #T GAG GAC ACG GCC GTG          336                                                                          Thr Ala Tyr Met Glu Leu His Ser Leu Arg Se - #r Glu Asp Thr Ala Val           #         90                                                                  - TAT TAC TGT GCG ACC TAC GGT CAT TAC TAC GG - #C TAC ATG TTT GCT TAC          384                                                                          Tyr Tyr Cys Ala Thr Tyr Gly His Tyr Tyr Gl - #y Tyr Met Phe Ala Tyr           #    105                                                                      - TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TC - #A GCC TCC ACC AAG GGC          432                                                                          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Se - #r Ala Ser Thr Lys Gly           110                 1 - #15                 1 - #20                 1 -       #25                                                                           #              433                                                            - (2) INFORMATION FOR SEQ ID NO:100:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 433 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 1..57                                                 #/note= "IDENTIFICATION METHOD: by                                            #with a known sequence or to an established                                   #sequence"     consensus                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 148..162                                              #/product= "hypervariable region 1"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 205..255                                              #/product= "hypervariable region 2"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 352..384                                              #/product= "hypervariable region 3"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                               - ATG GGA TGG AGC TGG ATC TTT CTC TTC CTC CT - #G TCA GGA ACT GCA GGT           48                                                                          Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Le - #u Ser Gly Thr Ala Gly           5                                                                             - GTC CTC TCT GAG GTG CAG CTG GTG CAG TCT GG - #A GCA GAG GTG AAG AAG           96                                                                          Val Leu Ser Glu Val Gln Leu Val Gln Ser Gl - #y Ala Glu Val Lys Lys           #           10                                                                - CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GC - #T TCC GGA TAC ACC TTC          144                                                                          Pro Gly Ala Ser Val Lys Val Ser Cys Lys Al - #a Ser Gly Tyr Thr Phe           #     25                                                                      - ACT GAC TAC AAC ATG GAC TGG GTG AAG CAG AG - #C CCT GGA AAG AGC CTC          192                                                                          Thr Asp Tyr Asn Met Asp Trp Val Lys Gln Se - #r Pro Gly Lys Ser Leu           # 45                                                                          - GAG TGG ATG GGA TAT ATT TAT CCT AAC AAT GG - #T GGT ACT GGC TAC AAC          240                                                                          Glu Trp Met Gly Tyr Ile Tyr Pro Asn Asn Gl - #y Gly Thr Gly Tyr Asn           #                 60                                                          - CAG AAG TTC AAG AGC AAG GCC ACC TTG ACC GT - #A GAC ACA TCC ACG AGC          288                                                                          Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Va - #l Asp Thr Ser Thr Ser           #             75                                                              - ACA GCC TAC ATG GAG CTG CAC AGC CTG AGA TC - #T GAG GAC ACG GCC GTG          336                                                                          Thr Ala Tyr Met Glu Leu His Ser Leu Arg Se - #r Glu Asp Thr Ala Val           #         90                                                                  - TAT TAC TGT GCG ACC TAC GGT CAT TAC TAC GG - #C TAC ATG TTT GCT TAC          384                                                                          Tyr Tyr Cys Ala Thr Tyr Gly His Tyr Tyr Gl - #y Tyr Met Phe Ala Tyr           #    105                                                                      - TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TC - #A GCC TCC ACC AAG GGC          432                                                                          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Se - #r Ala Ser Thr Lys Gly           110                 1 - #15                 1 - #20                 1 -       #25                                                                           #              433                                                            - (2) INFORMATION FOR SEQ ID NO:101:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 433 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 1..57                                                 #/note= "IDENTIFICATION METHOD: by                                            #with a known sequence or to an established                                   #sequence"     consensus                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 148..162                                              #/product= "hypervariable region 1"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 205..255                                              #/product= "hypervariable region 2"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 352..384                                              #/product= "hypervariable region 3"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                               - ATG GGA TGG AGC TGG ATC TTT CTC TTC CTC CT - #G TCA GGA ACT GCA GGT           48                                                                          Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Le - #u Ser Gly Thr Ala Gly           5                                                                             - GTC CTC TCT GAG GTG CAG CTG GTG CAG TCT GG - #A GCA GAG GTG AAG AAG           96                                                                          Val Leu Ser Glu Val Gln Leu Val Gln Ser Gl - #y Ala Glu Val Lys Lys           #           10                                                                - CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GC - #T TCC GGA TAC ACC TTC          144                                                                          Pro Gly Ala Ser Val Lys Val Ser Cys Lys Al - #a Ser Gly Tyr Thr Phe           #     25                                                                      - ACT GAC TAC AAC ATG GAC TGG GTG AAG CAG AG - #C CCT GGA CAA GGG CTC          192                                                                          Thr Asp Tyr Asn Met Asp Trp Val Lys Gln Se - #r Pro Gly Gln Gly Leu           # 45                                                                          - GAG TGG ATG GGA TAT ATT TAT CCT AAC AAT GG - #T GGT ACT GGC TAC AAC          240                                                                          Glu Trp Met Gly Tyr Ile Tyr Pro Asn Asn Gl - #y Gly Thr Gly Tyr Asn           #                 60                                                          - CAG AAG TTC AAG AGC AAG GCC ACC TTG ACC GT - #A GAC ACA TCC ACG AGC          288                                                                          Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Va - #l Asp Thr Ser Thr Ser           #             75                                                              - ACA GCC TAC ATG GAG CTG CAC AGC CTG AGA TC - #T GAG GAC ACG GCC GTG          336                                                                          Thr Ala Tyr Met Glu Leu His Ser Leu Arg Se - #r Glu Asp Thr Ala Val           #         90                                                                  - TAT TAC TGT GCG ACC TAC GGT CAT TAC TAC GG - #C TAC ATG TTT GCT TAC          384                                                                          Tyr Tyr Cys Ala Thr Tyr Gly His Tyr Tyr Gl - #y Tyr Met Phe Ala Tyr           #    105                                                                      - TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TC - #A GCC TCC ACC AAG GGC          432                                                                          Trp Gly Gln Gly Thr Leu Val Thr Val Ser Se - #r Ala Ser Thr Lys Gly           110                 1 - #15                 1 - #20                 1 -       #25                                                                           #              433                                                            - (2) INFORMATION FOR SEQ ID NO:102:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                               # 20               ACCA                                                       - (2) INFORMATION FOR SEQ ID NO:103:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                               # 20               TTCA                                                       - (2) INFORMATION FOR SEQ ID NO:104:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 390 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "synthetic DNA"RIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 1..66                                                 #/note= "IDENTIFICATION METHOD: by                                            #with a known sequence or to an established                                   #sequence"     consensus                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 136..165                                              #/product= "hypervariable region 1"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 211..231                                              #/product= "hypervariable region 2"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an estabished consensus sequence"                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: domain                                                          (B) LOCATION: 322..354                                              #/product= "hypervariable region 3"                                           #"IDENTIFICATION METHOD: by similarity with a known                           #or to an established consensus sequence"                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                               - ATG CAT TTT CAA GTG CAG ATT TTC AGC TTC CT - #G CTA ATC AGT GCC TCA           48                                                                          Met His Phe Gln Val Gln Ile Phe Ser Phe Le - #u Leu Ile Ser Ala Ser           10                                                                            - GTC ATA ATG TCC AGA GGA GAT ATC CAG CTG AC - #C CAG AGC CCA AGC AGC           96                                                                          Val Ile Met Ser Arg Gly Asp Ile Gln Leu Th - #r Gln Ser Pro Ser Ser           #   10                                                                        - CTG AGC GCT AGC CCA GGT GAC AGA GTG ACC AT - #C ACG TGC AGT GCC AGC          144                                                                          Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Il - #e Thr Cys Ser Ala Ser           #                 25                                                          - TCA AGT GTA AGT TAC ATG CAC TGG TTC CAG CA - #G AAA CCA GGT AAG GCT          192                                                                          Ser Ser Val Ser Tyr Met His Trp Phe Gln Gl - #n Lys Pro Gly Lys Ala           #             40                                                              - CCA AAG CTT TGG ATC TAC AGC ACA TCC AAC CT - #G GCT TCT GGT GTG CCA          240                                                                          Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Le - #u Ala Ser Gly Val Pro           #         55                                                                  - GCT AGA TTC AGC GGT AGC GGT AGC GGT ACA TC - #T TAC TCT CTC ACC ATC          288                                                                          Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Se - #r Tyr Ser Leu Thr Ile           #     70                                                                      - AGC CGA CTC CAG CCA GAG GAC ATC GCT ACA TA - #C TAC TGC CAG CAA AGG          336                                                                          Ser Arg Leu Gln Pro Glu Asp Ile Ala Thr Ty - #r Tyr Cys Gln Gln Arg           # 90                                                                          - AGT AGT TAC CCG TAC ACG TTC GGC GGG GGG AC - #C AAG GTG GAA ATC AAA          384                                                                          Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Th - #r Lys Val Glu Ile Lys           #                105                                                          #          390                                                                Arg Thr                                                                       __________________________________________________________________________

What is claimed is:
 1. A human CDR-grafted antibody containing aframework, FR, Kabat's Human Sub Group, HSG, which comprises an aminoacid sequence in which at least one amino acid of positions 38, 40, 67,72, 84 and 98 in the FR of H chain variable region and positions 4, 11,15, 35, 42, 46, 59, 69, 70, 71, 72, 76, 77 and 103 in the FR of L chainvariable region is replaced with other amino acid and a complementarydeterminant region, CDR, of a heavy chain has the amino acid sequencesas set forth in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 and a lightchain has the amino acid sequences as set forth in SEQ ID NO: 9, SEQ IDNO: 10 and SEQ ID NO:
 11. 2. The antibody as claimed in claim 1, whichbelongs to the class IgG.
 3. The antibody as claimed in claim 1, whereinthe H chain variable region has an amino acid sequence of SEQ ID NO:96and the L chain variable region has an amino acid sequence of SEQ IDNO:83.
 4. The antibody as claimed in claim 1, wherein the H chainvariable region has an amino acid sequence of SEQ ID NO:96 and the Lchain variable region has an amino acid sequence of SEQ ID NO:104. 5.The antibody as claimed in claim 3, which is produced by hybridoma FERMPB-5528.
 6. The antibody as claimed in claim 4, which is hybridoma FERMBP-5527.
 7. A composition comprising the antibody according to claim 1and a pharmaceutically acceptable carrier.
 8. A diagnostic compositioncomprising the antibody according to claim 1 as an active ingredient anda diagnostically acceptable carrier.
 9. The human CDR-grafted antibodyas claimed in claim 1, which comprises an amino acid sequence of the FRof the H chain and L chain variable regions of said human CDR-graftedantibody in which at least one amino acid is replaced with another aminoacid, and has antibody dependent cell mediated cytotoxic activity (ADCCactivity) which is at least the same as that of a human antibody havinga variable region of a monoclonal antibody obtained from a nonhumananimal which has been immunized with ganglioside GM₂.
 10. The humanCDR-grafted antibody as claimed in claim 1, which comprises a amino acidsequence of the FR of the H chain and L chain variable regions of saidhuman CDR-grafted antibody in which at least one amino acid is replacedwith another amino acid, and has complement dependent cytotoxic activity(CDC activity) which is at least the same as that of a human antibodyhaving a variable region of a monoclonal antibody obtained from anonhuman animal which has been immunized with ganglioside GM₂.